Optical analyses of particles and vesicles

ABSTRACT

This technology relates in part to optical methods for analyzing particles, including nanoparticles, thereby determining their presence, identity, origin, size and/or number in a sample of interest.

RELATED PATENT APPLICATION

This patent application claims the benefit of U.S. ProvisionalApplication No. 62/203,594, filed on Aug. 11, 2015, entitled OPTICALANALYSES OF PARTICLES AND VESICLES, naming John P. Nolan and ErikaDuggan as inventors, and designated by attorney docket no. CET-1001-PV.The entire content of the foregoing patent application is incorporatedherein by reference, including, without limitation, all text, tables anddrawings.

The subject matter claimed in this application was made with governmentsupport under Grant Number EB003824 awarded by the National Institutesof Health. The United States Government has certain rights in thissubject matter.

FIELD

The technology relates in part to optical methods for analyzingparticles and vesicles, including membrane vesicles such as liposomesand extracellular vesicles.

BACKGROUND

Optical methods for detecting particles and/or determining theiridentity, number, size or origin have long been in use. However, thestaining of particles using optically detectable labels generally mustbe accompanied by one or more washing and/or centrifugation proceduresto remove background interference from unbound label. Physicalseparation procedures, such as washing orcentrifugation/ultracentrifugation, can lead to inefficiencies as wellas inaccuracies in the analyses, especially when analyzing small volumesof sample, due to partial loss of particles during the separation.

In addition, the analyses of small particles, in the range of nanometersin diameter or less (e.g., about 100-200 nm or less), pose hurdles. Forexample, light scatter-based flow cytometry analyses of extracellularvesicles (EVs), exemplary of which are biological membrane vesicles thatare released from cell surfaces (ectosomes), internal stores (exosomes)or as a result of apoptosis or cell death, often provide incorrectestimates of their size and concentration when the vesicles arenanovesicles, due to dim light scatter. Further, detection of the EVsoften is triggered by coincidence, i.e., simultaneous detection of thepresence of more than one EV in the flow cytometer measurement volume,leading to incorrect concentration, size and fluorescence estimates.

Some optical methods, such as nanoparticle tracking analysis (NTA), alsoare limited in their ability to measure nanoparticles, due to theparticles scattering less light than the limits of detection. Inaddition, unlike flow cytometry, where the entire sample containing theparticles passes through the measurement volume, particles can diffusein and out of the probe volume during NTA measurements, resulting inover-counting of smaller particles and under-counting of largerparticles. Improved optical methods are needed for the detection ofparticles, including nanoparticles, among which are EVs, which often are500 nm or less in diameter.

SUMMARY

Provided in certain aspects is a method of analyzing particles in asample that includes: (a) contacting a sample comprising the particleswith one or more optically detectable labels, thereby forming a stainingsolution, where: (i) the one or more optically detectable labels includea surface area probe or volume probe, where the surface area probeinteracts with the particles stoichiometrically with respect to particlesurface area or the volume probe interacts with the particlesstoichiometrically with respect to particle volume, thereby formingparticles that include particle-associated surface area probe or volumeprobe, where the optical signal from the particle-associated surfacearea probe or volume probe is proportional to the surface area or volumeof the particle, respectively, and/or (ii) the one or more opticallydetectable labels include a molecular marker-specific probe, where themolecular marker-specific probe interacts with a molecular marker of theparticle stoichiometrically with respect to the number of molecules ofthe molecular marker that are associated with the particle, therebyforming particles that include particle-associated molecularmarker-specific probe, where the optical signal from theparticle-associated molecular marker-specific probe is proportional tothe number of molecules of molecular marker associated with theparticle; and (b) without physical separation or isolation of theparticles, detecting the optical signal of the one or moreparticle-associated optically detectable labels generated in (i) and/or(ii), thereby analyzing the particles in the sample.

Also provided in certain aspects is a method of detecting, identifying,quantifying and/or determining the size of at least a first particlespecies in a sample that includes at two distinct particle species by:(a) contacting a sample containing at least two distinct particlespecies, where the distinct particle species differ from one another bysize and/or by least one molecular marker associated with each particlespecies, with one or more optically detectable labels comprising asurface area probe or volume probe, where the surface area probe orvolume probe interacts with at least a first particle speciesstoichiometrically with respect to particle surface area or volume,respectively, thereby forming particles that include particle-associatedsurface area probe or volume probe, where the optical signal from theparticle-associated surface area probe or volume probe is proportionalto the surface area or volume, respectively, of the first particlespecies; and/or (b) contacting the sample with one or more opticallydetectable labels that include a molecular marker-specific probe, wherethe molecular marker-specific probe interacts with a molecular marker ofat least the first particle species stoichiometrically with respect tothe number of molecules of the molecular marker that are associated withthe particle, thereby forming particles that include particle-associatedmolecular marker-specific probe, where the optical signal from theparticle-associated molecular marker-specific probe is proportional tothe number of molecules of the molecular marker that are associated withthe first particle species; (c) detecting an optical signal from theparticle-associated surface area probe or volume probe and/or detectingan optical signal from the particle-associated molecular marker-specificprobe, thereby obtaining an optical signal intensity from theparticle-associated surface area probe or volume probe and/or theparticle-associated molecular marker-specific probe; (d) based on theoptical intensity of the particle-associated surface area probe orvolume probe obtained in (c), determining the surface area or volume ofat least the first particle species, thereby detecting and/ordetermining the size of at least the first particle species in thesample; and/or (e) based on the optical intensity of theparticle-associated molecular marker-specific probe obtained in (c),determining the type and/or number of molecular markers associated withat least the first particle species, thereby detecting, identifyingand/or quantifying at least the first particle species in the sample. Incertain aspects of the method, the particle species are nanoparticlespecies.

The terms “associated,” “associated with” or “interact,” as used hereininterchangeably with “bound” or “containing,” e.g., “lipid-containingparticle,” can refer to a variety of different types of contact between,for example, a particle and its components (lipids, proteins, nucleicacids, carbohydrates, glycoproteins, glycolipids, phospholipids,phosphosphingolipids, etc.) or between a particle and an opticallydetectable label that can include, but is not limited to, covalent bondsor non-covalent interactions, non-limiting examples of which include vander Waals interactions, hydrogen bonding, ionic interactions,electrostatic interactions and/or hydrophilic or hydrophobicinteractions. In embodiments, the molecule that is the probe is also anoptically detectable label, e.g., di-8-ANEPPS.

With respect to the interaction of membrane vesicles, liposomes,extracellular vesicles and other lipid bilayer or lipid membranecontaining particles, the terms “associated,” “associated with” or“interact,” as used herein, also can refer to intercalation of theoptically detectable label into the membrane, or binding of theoptically detectable label to a molecular marker within or at thesurface of the membrane vesicles, liposomes, extracellular vesicles andother lipid bilayer or lipid membrane containing particles. The term“free” or “unbound,” as used herein, refers to molecules, includingoptically detectable labels, which are not in contact with the particle.“Free” or “unbound” optically detectable label, e.g., in the stainingsolution, generally is detected as a background signal or no signal,relative to the higher signal intensity of the optically detectablelabel when it is associated with a particle (i.e., a particle-associatedsurface area probe or volume probe, or a particle-associated molecularmarker-specific probe).

Any particle in the size range of nm to microns or larger can beanalyzed according to the methods provided herein. In certain aspects,the particle is a nanoparticle of less than 1 micron in diameter. Inaspects of the methods provided herein, the nanoparticles in the sampleinclude at least one particle with a size of about 500 nm or less indiameter, between about 10 nm to about 200 nm in diameter, between about50 nm to about 200 nm in diameter, between about 50 nm to about 150 nmin diameter, between about 10 nm to about 500 nm in diameter, betweenabout 50 nm to about 200 nm in diameter, or between about 50 nm to about150 nm in diameter.

In certain aspects, the concentration of the particles in the sample isadjusted so the particle is optimally stained with, or associated with,or bound to, the optically detectable label. In some aspects, theparticle concentration can be adjusted to between about 1×10³particles/μL to about 1×10¹⁵ particles/μL; between about 1×10⁴particles/μL to about 1×10¹⁴ particles/μL; between about 1×10⁵particles/μL to about 1×10¹³ particles/μL; between about 1×10⁴particles/μL to about 1×10¹² particles/μL; between about 1×10⁶particles/μL to about 1×10¹² particles/μL; between about 1×10⁶particles/μL to about 1×10¹¹ particles/μL; between about 1×10⁶particles/μL to about 1 ×10¹⁰ particles/μL; between about 1×10⁷particles/μL to about 1×10¹⁰ particles/μL, between about 1×10⁸particles/μL to about 1×10¹⁹ particles/μL; or about 1×10⁹ particles/μL.

In certain aspects, the concentration of the particles in the sample isadjusted using a suitable buffer, such as an isotonic buffer, wherebythe resulting staining solution contains a buffer. In aspects, thestaining solution includes a surfactant, or a mixture of surfactants.Without being bound by theory, the surfactant could, in someembodiments, facilitate staining of the particles in the stainingsolution, such as the lipid bilayers of membrane vesicles, liposomes orextracellular vesicles. In some aspects, the surfactant can be added tothe staining solution in an amount of between about 0.001% to about0.5%; between about 0.002% to about 0.4%; between about 0.003% to about0.3%;

between about 0.004% to about 0.2%; between about 0.001% to about 0.1%;between about 0.005% to about 0.05%; between about 0.005% to about0.04%; between about 0.005% to about 0.02%; or about 0.01%. In aspectsof the methods provided herein, the surfactant can be a nonionicpoloxamer, such as the Synperonics, Pluronics and Kolliphor classes ofpoloxamers. In some aspects, the surfactant can be a Pluronic poloxamer.In aspects, the Pluronic poloxamer can be Pluronic-127.

In aspects of the methods provided herein, analyzing the particles inthe sample can include detecting the particles in the sample. Ingeneral, “analyzing the particles,” as used herein, refers to thedetection and analysis of individual particles in the sample, such as byflow cytometry. As used herein analyzing the particles “in bulk” meansthat the particles are analyzed as a whole, without resolution of theindividual particles from one another, such as, for example, measuringthe absorbance of a suspension of particles in a cuvette using afluorimeter. A bulk analysis can be distinguished, for example, from thedetection and analysis of individual particles, such as by flowcytometry. In certain embodiments, a bulk analysis also can include thedetection and analysis of individual particles without distinguishingthe individual particles from one another, such as identifying EVs in asample without distinguishing them according to the cells from whichthey are derived and/or signature markers associated with different EVs.In certain aspects, analyzing the particles in the sample can includedetermining the surface area or volume of the particle based on thedetected optical signal of the particle-associated surface area probe orvolume probe, respectively. In some aspects, the size of the particlecan be determined based on the surface area or volume. In aspects,determining the size of the particle includes determining the diameterof the particle.

In some aspects of the methods provided herein, analyzing the particlesin the sample can include determining the type and/or number ofmolecular markers associated with the particle based on the detectedoptical signal of the molecular marker-associated probe. In aspects, theparticle can be identified and/or quantified based on the type and/ornumber of molecular markers associated with the particle.

In certain aspects of the methods provided herein, the surface areaprobe or volume probe is a fluorescent label. In some aspects, themolecular marker-specific probe is a fluorescent label. Any fluorescentlabel can be used in the methods provided herein including, but notlimited to, a fluorophore, a tandem conjugate between more than onefluorophore, a fluorescent polymer, a fluorescent protein, or afluorophore conjugated to a molecule that interacts with one or moreparticles of the sample. In some aspects, the molecule that interactswith one or more particles of the sample includes, but is not limitedto, a protein, an antibody, a lectin, a peptide, a nucleic acid, acarbohydrate or a glycan. The molecule can interact with the particle ina manner that is proportional to the surface area or volume of theparticle, or can bind or otherwise associate specifically with one ormore molecular markers on the particle.

In certain aspects of the methods provided herein, the molecule thatinteracts with one or more particles of the sample is an antibody, or amolecular marker-binding/associating fragment thereof. Antibodies bindto specific antigens and contain two identical heavy chains and twoidentical light chains covalently linked by disulfide bonds. Both theheavy and light chains contain variable regions, which bind the antigen,and constant (C) regions. In each chain, one domain (V) has a variableamino acid sequence depending on the antibody specificity of themolecule. The other domain (C) has a rather constant sequence commonamong molecules of the same class. The domains are numbered in sequencefrom the amino-terminal end. For example, the IgG light chain includestwo immunoglobulin domains linked from N- to C-terminus in the orderV_(L)-C_(L), referring to the light chain variable domain and the lightchain constant domain, respectively. The IgG heavy chain includes fourimmunoglobulin domains linked from the N- to C-terminus in the orderV_(H)-C_(H)1-C_(H)2-C_(H)3, referring to the variable heavy domain,contain heavy domain 1, constant heavy domain 2, and constant heavydomain 3. The resulting antibody molecule is a four chain molecule whereeach heavy chain is linked to a light chain by a disulfide bond, and thetwo heavy chains are linked to each other by disulfide bonds. Linkage ofthe heavy chains is mediated by a flexible region of the heavy chain,known as the hinge region. Fragments of antibody molecules can begenerated, such as for example, by enzymatic cleavage. For example, uponprotease cleavage by papain, a dimer of the heavy chain constantregions, the Fc domain, is cleaved from the two Fab regions (i.e. theportions containing the variable regions).

In humans, there are five antibody isotypes classified based on theirheavy chains denoted as delta (δ), gamma (γ), mu (μ), and alpha (α) andepsilon (ε), giving rise to the IgD, IgG, IgM, IgA, and IgE classes ofantibodies, respectively. The IgA and IgG classes contain the subclassesIgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Any such antibody that is fulllength or a portion thereof that is less than full length, e.g.,containing a heavy chain, light chain, Fab, Fab₂, Fv, or Fc, iscontemplated for use in the methods herein. In some aspects, the portionof an antibody can be a single chain variable fragment (scFv) of anantibody. In some embodiments, the antibody is a camelid single domainantibody. In certain aspects, the antibody or portion thereof isconjugated to a fluorophore. In aspects, the antibody is selected fromamong anti-CD61, anti-CD171, anti-CD325, anti-CD130, anti-GLAST,anti-EGFRvIII, anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9,anti-CD41, anti-CD235, anti-CD54, anti-CD45 and anti-IgG. In someaspects, the fluorophore is selected from among DyLight488, a BrilliantViolet dye (exemplary of which are BV-421, BV-510, BV-605 and the like),Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, FITC, PE-Cy5.5,PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.

In certain aspects of the methods provided herein, at least one particleof the sample includes a lipid bilayer. In aspects, the particlecontaining a lipid bilayer can be a membrane vesicle, a lipoprotein, aliposome or an extracellular vesicle.

The optically detectable labels associated with the particles in thesamples analyzed according to the methods provided herein can bedetected using a number of methods including, but not limited to, visualinspection, microscopy, spectroscopy, fluorescence spectroscopy,fluorescence imaging, imaging flow cytometry or flow cytometry. Incertain aspects, the detection is by flow cytometry and the samples areanalyzed by flow cytometry.

In aspects, the optically detectable labels used in the analysis by flowcytometry are fluorescent labels. In some aspects, one or more of theparticles analyzed according to the methods provided herein includesmembrane vesicles, lipoproteins, liposomes, extracellular vesicles orother particles containing a lipid bilayer membrane, or combinationsthereof. In aspects, the surface area probe or volume probe thatinteracts with the particle containing a lipid bilayer membrane isselected from among di-8-ANEPPS, di-4-ANEPPS, F2N12S, FM-143, Cell MaskOrange, Cell Mask Green, Cell Mask Deep Red, a carbocyanine dye or a PKHdye. In some aspects, the surface area probe or volume probeintercalates into the bilayer membrane. In aspects, the surface areaprobe is di-8-ANEPPS.

In certain aspects of the methods provided herein, the surface areaprobe or volume probe is added in an amount such that the ratio of theamount surface area probe or volume probe (P) relative to the amount oflipid (L) in the particle, P/L, is adjusted whereby the surface areaprobe or volume probe interacts with the particles stoichiometricallywith respect to particle surface area or volume, respectively. In someaspects, the P/L ratio is between about 0.1 to about 0.25.

In some aspects of the methods provided herein, the molecularmarker-specific probe is a fluorophore conjugated to a protein. In someaspects, the protein is selected from among annexin V, cholera toxinB-subunit, anti-CD61, anti-CD171, anti-CD325, anti-CD130, anti-GLAST,anti-EGFRvIII, anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9,anti-CD41, anti-CD235, anti-CD54 and anti-CD45. In certain aspects, thefluorophore conjugated to the protein conjugates is selected from amongDylight488, a Brilliant Violet dye, Pacific Blue, Chrome Orange,Brilliant Blue 515, PE, rhodamine, FITC, PE-Cy5.5, PE-Cy7, APC,Alexa647, APC-Alexa700 and APC-Alexa750.

In aspects of the methods provided herein, physical separation orisolation of the particles includes filtration, washing the particles orprecipitating the particles out of the sample or solution containing theparticles. In some aspects, physical separation or isolation of theparticles includes centrifugation or ultracentrifugation of theparticles.

In aspects of the methods provided herein, the flow cytometer has aconfiguration whereby light is collected from one side of the flow cell.In some aspects, the flow cytometer has a configuration whereby light iscollected from both sides of the flow cell. In certain aspects, thedetection range of the flow cytometer is between about 1 fluorescentmolecule per particle to about 5, 10, 15, 20, 30, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 or morefluorescent molecules per particle. In some aspects, the resolutionthreshold of the flow cytometer is less than 200 fluorescent moleculesper particle. In aspects, the resolution threshold of the flow cytometeris between about 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 fluorescent molecules per particle to about 50, 100 or150 fluorescent molecules per particle.

In certain aspects of the methods provided herein, the particle is anextracellular vesicle, and, based on the detected optical signal of themolecular marker-specific probe, the type of molecular marker associatedwith the extracellular vesicle is determined. In some aspects, the celland/or tissue of origin of the extracellular vesicle is identified basedon the type of molecular marker associated with the extracellularvesicle.

In some aspects of the methods provided herein, one or more opticalstandard particles can be used to provide improved accuracy indetermining the optical intensity of the optically detectable labelsassociated with the particles. In certain aspects, the optical standardparticle can be a particle whose surface area or volume or diameter ispredetermined by a method that does not use an optically detectablelabel, such as NTA, tunable resistive pulse sensing (TRPS), electronmicroscopy (EM) or other methods. In aspects, the optical standardparticle is capable of binding to or otherwise associating with anoptically detectable label that is a surface area probe or a volumeprobe. The optical standard particle can then be contacted with anoptically detectable label that is a surface area probe or volume probeand the intensity of the label associated with the optical standardparticle obtained, thereby providing a correlation between surface areaor volume, respectively, and optical intensity.

In some aspects, the optical standard particle is a particle containingmolecular marker molecules that can be bound to or otherwise associatedwith one or more optically detectable labels that are molecularmarker-specific probes. The optical intensity of the molecularmarker-specific probe-associated optical standard particle can bestandardized against the measured optical intensity of a known externalstandard, thereby providing a correlation between optical intensity andthe number of molecules of molecular marker associated with a particle.

In certain aspects, the optical standard particle is a liposome or otherlipid-containing particle. In aspects, the amount of lipid in thelipid-containing optical standard particle is known. In some aspects,the optical standard particle is a silica particle. In aspects, thesilica particle includes a lipid bilayer. In some aspects, the opticalstandard particle is a bead. In some aspects, the bead can captureligands that can bind to one or more molecular markers associated with aparticle. In aspects, the ligand is an antibody. In certain aspects, theligand is conjugated to an optically detectable label.

In some aspects, the optical standard particle is in a collection orpreparation of optical standard particles that include a sizedistribution of optical reference particles, whereby a regressioncorrelation between a distribution of sizes/surface area and opticalintensities of the optical standard particles associated with anoptically detectable label can be obtained. In aspects, the opticalstandard particle is in a collection or preparation of optical standardparticles that include a distribution of numbers of molecular markersassociated with each particle in the preparation, whereby a regressioncorrelation between a distribution of numbers of molecules of molecularmarker per optical standard particle and the optical intensities of theoptical standard particles associated with an optically detectable labelcan be obtained.

In certain aspects, the optical standard particles can be used for theanalysis of particles according to the methods provided herein. In someaspects, the analysis is by flow cytometry. In aspects, the opticalstandard particle is a liposome, or a silica particle that includes alipid bilayer. In some aspects, the optically detectable labelassociated with the liposome or the lipid bilayer of the silica particleis di-8-ANEPPS or fluorescently labeled (e.g., with DyLight488) annexinV. In certain aspects, the optical standard particle is a bead that canbind to or otherwise associate with a ligand.

In aspects, the ligand is an antibody. Any antibody as known and asdescribed herein with respect to any aspect of the methods providedherein can be used as a ligand. In some aspects, the antibody is labeledwith a fluorophore. In aspects, the antibody is selected from amonganti-CD61, anti-CD171, anti-CD325, anti-CD130, anti-GLAST,anti-EGFRvIII, anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9,anti-CD41, anti-CD235, anti-CD54, anti-CD45 and anti-IgG. In someaspects, the fluorophore is selected from among DyLight488, a BrilliantViolet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515, PE, FITC,PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 and APC-Alexa750.

In certain aspects, the methods provided herein are for simultaneouslyanalyzing a plurality of particles of different size and/or havingdifferent molecular markers. In some aspects, the different molecularmarkers can simultaneously be detected according to the methods providedherein, using optically detectable labels that are distinct from oneanother for each of the different molecular markers. In aspects, formultispectral analysis of a plurality of particles having a plurality ofmolecular markers, provided herein is a panel of optical standardparticles, each associated with a distinct molecular marker conjugatedto a distinct optically detectable label whereby, based on the measuredoptical intensities of the panel of optical standard particles, theoptical intensities of the corresponding optically detectable labelsassociated with the molecular markers of the particles are measured withimproved accuracy (e.g., by facilitating “spectral unmixing”). In someaspects, the analysis is by flow cytometry. In aspects, the panel ofoptical standard particles includes fluorescent beads. In some aspects,the panel of optical standard particles includes beads that can bind toor otherwise associate with ligands, which in turn can be labeled with afluorophore. In aspects, the ligand is an antibody. In aspects, theantibody is selected from among anti-CD61, anti-CD171, anti-CD325,anti-CD130, anti-GLAST, anti-EGFRvIII, anti-EGFR, anti-CD133, anti-CD15,anti-CD63, anti-CD9, anti-CD41, anti-CD235, anti-CD54, anti-CD45 andanti-IgG. In some aspects, the fluorophore is selected from amongDyLight488, a Brilliant Violet dye, Pacific Blue, Chrome Orange,Brilliant Blue 515, PE, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647,APC-Alexa700 and APC-Alexa750.

The size of an optical standard particle for use in the methods providedherein can be between about 20 nm to about 1,2, 3,4, 5, 10 or moremicrons. In some aspects, the size of the optical standard particle isbetween about 20 nm to about 5 microns, about 30 nm to about 3 microns,about 40 nm to about 2 microns, about 50 nm to about 1 micron, about 50nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about400 nm, about 100 nm to about 450 nm, or about 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490 or 500 nm.

In aspects of the methods provided herein, an optical standard particlethat is not associated with an optically detectable label can be used,thereby improving accuracy by correcting the background optical signalobtained from the particle alone. In certain aspects, the opticalstandard particle is a bead. In aspects, the bead is coated with, boundto, or otherwise associated with a molecule. In some aspects, themolecule is a polymer. In certain aspects, the polymer is polyethyleneglycol (PEG). In aspects, the polymer is a protein that does notassociate with an optically detectable label. In certain aspects, theprotein is BSA.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIGS. 1A to 1F show the detection of fluorescently labeled particles byflow cytometry using a fluorescent trigger (FIGS. 1A to 1C) or a sidescatter trigger (FIGS. 1D to 1F). FIG. 1A and FIG. 1D depict the 488 SSCpeaks corresponding to 0.53 μm and 0.11 μm fluorescent beads. FIG. 1Band FIG. 1E depict the Nile Red fluorescence peaks corresponding to 0.53μm and 0.11 μm fluorescent beads. FIG. 1C and FIG. 1F depict a plot ofthe 488 SSC peaks against the Nile Red fluorescence peaks.

FIGS. 2A to 2H show a comparison of size distribution profiles ofvesicles using fluorescence intensity histograms obtained by flowcytometry (FIGS. 2A to 2D) or using nanoparticle diameter populationhistograms obtained by nanoparticle tracking analysis (NTA) (FIGS. 2E to2H). FIG. 2A and FIG. 2E depict fluorescence intensity histograms ofdi-8-ANEPPS stained vesicles prepared by extrusion through polycarbonatemembrane filters with average pore sizes of 200 nm. FIG. 2B and FIG. 2Fdepict fluorescence intensity histograms of di-8-ANEPPS stained vesiclesprepared by extrusion through polycarbonate membrane filters withaverage pore sizes of 100 nm. FIG. 2C and FIG. 2G depict fluorescenceintensity histograms of di-8-ANEPPS stained vesicles prepared byextrusion through polycarbonate membrane filters with average pore sizesof 80 nm. FIG. 2D and FIG. 2H depict fluorescence intensity histogramsof di-8-ANEPPS stained vesicles prepared by extrusion throughpolycarbonate membrane filters with average pore sizes of 50 nm.

FIG. 3 is a graph depicting the relationship between fluorescenceintensity of a fluorescent probe associated with vesicles, and vesiclesurface area.

FIGS. 4A to 4F show the measurement of extracellular vesicles (EVs) inrat plasma by NTA (FIGS. 4A to 4C) or fluorescence triggered flowcytometry (FIGS. 4D to 4F), with their diameter calibrated usingsynthetic liposomes as reference particles. FIG. 4A, FIG. 4B and FIG. 4Cdepict nanoparticle population size distributions of three different ratplasma samples using NTA. FIG. 4D, FIG. 4E and FIG. 4F depictnanoparticle population size distributions of the three different ratplasma samples using flow cytometry. FIG. 4G shows plasma nanoparticle(EV) concentrations as measured by NTA and flow cytometry for eightanimals.

FIGS. 5A to 5D show the measurement of surface molecular markers of EVsin plasma from control plasma or ionophore-treated platelet rich plasmastained with di-8-ANEPPS and DyLight488-Annexin V orDylight488-anti-CD61. FIG. 5A shows the measurement of surface molecularmarkers of EVs in control plasma stained with di-8-ANEPPS andDyLight488-Annexin V. FIG. 5B shows the measurement of surface molecularmarkers of EVs in control plasma stained with di-8-ANEPPS andDyLight488-anti-CD61. FIG. 5C shows the measurement of surface molecularmarkers of EVs in ionophore-treated platelet rich plasma stained withdi-8-ANEPPS and DyLight488-Annexin V. FIG. 5D shows the measurement ofsurface molecular markers of EVs in ionophore-treated platelet richplasma stained with di-8-ANEPPS and DyLight488-anti-CD61.

FIG. 6 depicts fluorescence staining of polystyrene antibody capturebeads coated with anti-lambda IgG and stained with a DyLight 488conjugated antibody.

FIG. 7 depicts specific fluorescence staining of synthetic liposomescontaining phosphatidylserine (PS) in a population containing a mixtureof synthetic liposomes that contain or do not contain PS.

FIG. 8 shows fluorescence staining of silica spheres coated with a lipidbilayer.

FIGS. 9A to 9F show measurement of a fluorescence spectral shift tomeasure saturation of a lipid-containing particle using a membrane dye.FIGS. 9A to 9E depict measurements performed on synthetic liposomeshaving known amounts of associated lipid, and FIG. 9F depictsmeasurements performed on a sample of platelet-poor plasma (PPP). FIG.9A is a fluorescence spectrum of bulk suspensions of di-8-ANEPPS (500nM) in buffer alone (HBS; 150 mM NaCl, 10 mM HEPES pH 7.4) or bufferplus two concentrations of synthetic lipid vesicles (50 uM and 3 uM).FIG. 9B is a normalized representation of the measurements depicted inFIG. 9A. FIG. 9C depicts the ratio of intensities at 690 to 610 nmmeasured at several different probe to lipid ratios. FIG. 9D depictshistograms of the population distributions of the ratio of intensitiesof the synthetic vesicles measured through the 690/50 nm and 610/20 nmfilters (690/610 ratio), for high (0.16) and low (0.01) probe to lipidratios. FIG. 9E depicts the ratio of intensities at 690 to 610 nmmeasured by flow cytometry of synthetic vesicle preparations havingseveral different probe to lipid ratios. FIG. 9F depicts the median690/610 ratio at two dilutions of human PPP.

FIGS. 10A to 10E depict measurement of light scatter in samplescontaining vesicles, using a fluorescence-based detection approach. FIG.10A depicts buffer alone, FIG. 10B depicts buffer+probe, FIG. 100depicts a sample preparation containing synthetic vesicles stained withprobe, FIG. 10D depicts the sample preparation of FIG. 100 with addeddetergent (Triton X-100; TX100), FIG. 10E depicts a platelet-free plasmapreparation stained with dye, and FIG. 10F depicts the samplepreparation of FIG. 10E with added detergent (Triton X-100; TX100).

FIGS. 11A to 11D depict the analysis of EVs in human plasma usingmultiple markers. FIG. 11A depicts measurements performed on syntheticliposomes, FIG. 11B depicts measurements performed on syntheticliposomes to which detergent is added (Triton X-100; TX100), FIG. 110depicts measurements performed on platelet-rich plasma (PRP) supernatantand FIG. 11D depicts measurements performed on platelet-rich plasma(PRP) supernatant to which detergent is added (Triton X-100; TX100).

DETAILED DESCRIPTION

Provided herein are optical methods for analyzing particles or vesicleswith improved efficiency and accuracy. The methods provided herein canbe used to analyze particles or vesicles of size ranging from about 1 nmin diameter to 100 microns (μm) or more in diameter. The analysis caninclude, but is not limited to, detection, quantitation, sizing andcharacterization of the particles, which can include determining theidentity, i.e., molecular content and origin of the particles (e.g.,cell/tissue of origin of an extracellular vesicle). The improvedefficiency and accuracy of the methods provided herein permits theanalysis of a wider range of particle sizes, including nanoparticles ornanovesicles of about 200 nm or less in diameter.

Overview of the Methods

Exemplary aspects of the methods provided herein are now described.Samples containing particles of interest, including microparticles,nanoparticles, liposomes, vesicles (unilamellar, multilamellar, e.g.),lipoproteins, endosomes, viruses, viral particles, virus-like particles,apoptotic bodies and/or extracellular vesicles (EVs), are either at aparticle concentration or are diluted to a sample particle concentrationthat facilitates optimal staining with an optically detectable label,detection of the label and analysis. For example, when the analysis isby flow cytometry, the samples are at, or can be diluted to, a finalparticle concentration of about 1×10⁸ to about 1×10¹⁰ particles/μl.

The optimal dilution can, in embodiments, be determined by serialdilution of the sample in the presence of a constant amount of opticallydetectable label, thereby determining the optimal dilution (particleconcentration) for enhanced signal from the label associated with theparticles and low to negligible background signal from the unbound orfree label. The particle concentration of the diluted sample thatproduces optimal enhanced signal relative to background noise canindependently be measured by a technique not involving contact with theoptically detectable label, such as nanoparticle tracking analysis(NTA), transmission electron microscopy (TEM) or resistive pulsespectroscopy (RPS), to determine the equivalent particle concentrationof the optimally diluted sample.

For example, when the sample is plasma, the dilution factor foranalyzing membrane vesicles (e.g., EVs) in the plasma is high due to thepresence of high concentrations of proteins that can non-specificallycompete with the vesicles for binding/association of the opticallydetectable label. In such instances, when the sample is plasma, thedilution factor can be of the order of between 100-fold to 200-fold, oreven 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 20,000,30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000-fold ormore. When the sample is cerebrospinal fluid, which has lower amounts ofprotein, the dilution factor for the analysis of membrane vesicles inthe fluid can be lower, of the order of, for example, 20-fold, or in therange of between 5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100-fold or higher. In general, depending on thesample, the dilution factor can be anywhere from about 2-fold to about5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000,100,000-fold or higher. In certain embodiments, no dilution of thesample may be needed.

In some embodiments, the sample can be treated to remove, in whole or inpart, matter other than the particles such as undesired largeparticulates, cells, cellular debirs or other undissolved subject matterthat does not include the particles. For example, to remove largeparticulates or cellular debris from a biological sample such as bloodor plasma or cerebrospinal fluid, the sample can be subjected tocentrifugation at 2500 g for one, two, three or more times, each step ofcentrifugation being performed for about 1 minute to about 20 minutes ormore, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 or more minutes. In some embodiments, thecentrifugation is performed two times for about 10 minutes each at 2500g. In embodiments, the centrifugation is performed prior to dilution foroptimal staining with an optically detectable label.

The sample containing an adjusted particle concentration as describedabove can be stained using one or more optically detectable labels. Theoptically detectable labels and/or staining conditions are selected suchthat binding or other association of the labels to the particles isstoichiometric and/or saturable with respect to one or more of: (a) thesurface area or volume of the particle; or (b) one or more specificmolecular markers to which the optically detectable labels are bound,whereby the optical signal from the label is proportional to the surfacearea/volume and/or number of molecular markers of the particle, therebyproviding information about the size, features and/or origin of theparticle. The staining can be performed before, after orcontemporaneously with the sample dilution. The optically detectablelabel can, in some embodiments, be a probe that can intercalate into theparticle stoichiometrically with respect to the surface area and/orvolume of the particle, thereby producing an optical signal that isproportional to the surface area and/or volume of the particle. Forexample, in particles that are lipid vesicles (e.g., liposomes or EVs),the fluorescent label di-8-ANEPPS(442-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]1-(3-sulfopropyl)-pyridinium)can bind to lipid membranes in a stoichiometric manner that isproportional to the surface area or volume of the liposomes. Anexemplary volume probe for use in any of the methods herein iscarboxyfluorescein succinimidyl ester (CFSE).

In certain embodiments, the optically detectable label specificallybinds or otherwise associates stoichiometrically with respect to one ormore molecular components/markers of the particle (molecularmarker-specific probe), thereby providing an optical signal that isspecific for the marker and permits identification of the type ofparticle based on the type of detected marker. In embodiments, themolecular marker-specific probe binds or otherwise associates with themolecular marker in a stoichiometric manner proportional to the numberof molecules of molecular marker per particle. As used herein, a“molecular marker” is a molecule that is a specific component or ligandof a particular type of particle. The molecular marker can be presentanywhere in the interior or on the surface of the particle, or can beassociated with the membrane when particle is a vesicle (e.g., membranevesicles, liposomes, EVs), and detection of the molecular marker canidentify the type of particle associated with the molecular marker. Insome embodiments, the molecular marker can be present on the surface ofthe particle. For example, in particles that are lipid vesicles (e.g.,liposomes or EVs), annexin V has a specific binding affinity forphosphatidyl serine (PS), which is a surface molecular marker of manycell-derived EVs, membrane vesicles and liposomes. The number ofcell-derived EVs or other PS-containing vesicles can be determined bystaining with an optically labeled annexin V, e.g., annexin V conjugatedto the fluorescent label, Dylight488-succinimidyl ester. As anotherexample, platelet-derived extracellular vesicles (EVs) have CD61 as amolecular marker, which can be detected using anti-CD61 that has beenlabeled with an optically detectable label. Identifying the type ofparticle can include identifying its origin or source. For example, whenthe particle is an EV, as indicated in the aforementioned example, thedetection of CD61 in the EV can identify the EV as originating fromplatelets.

In some embodiments, the particles can be stained with both a surfacearea probe or volume probe for optical detection, and a molecularmarker-specific optical label. The concentration of the opticallydetectable label, the choice of staining buffer, the temperature duringstaining and/or the staining time can be adjusted to achievestoichiometric incorporation of the optically detectable label in theinterior and/or surface of the particle. Any optically detectable labelthat shows enhanced intensity (e.g., color, fluorescence, luminescence,bioluminescence, chemiluminescence and light scatter) when bound to theparticles relative to the free label can be used in the methods providedherein.

The term “stoichiometric,” as used herein, refers to the association orbinding of a surface area/volume probe or molecular marker-specificprobe to a particle in a manner that is proportional to the surfacearea/volume of a particle (for a surface area/volume probe) or thenumber or concentration of molecules of molecular marker-specific probeassociated with the particle, whereby, based on the intensity of thesignal generated from the optically detectable label associated with theprobe, the surface area of the particle or the number/concentration ofmolecules of marker-specific probe, respectively, can be determined.

The terms “saturable,” “saturated,’ “saturating,” “approachingsaturation,” as used herein, refer to the amount of probe (surface areaor molecular-marker specific) which, when associated with or bound to aparticle, generates a signal from an optically detectable labelassociated with the probe that does not substantially increase when moreprobe is added to the sample containing the particle. For example, whenthe optically detectable label associated with the probe is bound to theparticle in a saturating amount, adding further amounts of the probedoes not substantially increase the signal generated by the opticallydetectable label by more than 0% 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.75. 0.8%. 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%,5.0%, 5.5%, 6%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0% or more,up to 15% or 20%. In embodiments of the methods provided herein,stoichiometric binding/association of the surface probe or molecularmarker-specific probe to a particle is achieved when thebinding/association of the probe to/with the particle is in an amountthat is saturating for the signal intensity of the optically detectablelabel associated with the probe. In some embodiments of the methodsprovided herein, the optically detectable label used in the methods issaturable when bound/associated as a probe to a particle. For example,di-8-ANEPPS is a saturable optically detectable surface area probe.

In lipid-containing particles such as liposomes, EVs or otherlipid-containing vesicles, when probe is added to such particles, theprobe often becomes associated with the particles by intercalation intothe lipid bilayer membranes. In such embodiments, as more probe is addedto the particles, the optimal probe to lipid ratio is considered as“approaching saturation” rather than becoming saturated becausesaturation can lead to self-quenching among the large number ofintercalated probe molecules. Thus, as used herein a probe amount“approaching saturation” or that “approaches saturation” can be usedinterchangeably with “saturable,” “saturated,’ “saturating” and the likeand refers to the amount of probe (surface area or molecular-markerspecific) which, when associated with or bound to a particle, generatesa signal from an optically detectable label associated with the probethat does not substantially increase when more probe is added to thesample containing the particle.

In some aspects, for analysis of the stained particles, the methodsprovided herein do not include a physical separation or isolation. Asused herein, “physical separation or isolation” means that thenon-particle reaction components of the staining reaction aresubstantially removed from the presence of the stained particles (e.g.,the container in which the stained particles are present) by methodssuch as filtration, precipitation, washing or centrifugation, includingultracentrifugation. As used herein, “non-particle reaction components”or “non-particle components” of the staining reactions refers to allcomponents of the staining reaction other than the particles, some orall of which include bound (particle-associated) optically detectablelabel; the non-particle components can include buffers, salts,surfactants, unbound (free) optically detectable label and othercomponents that are present in the staining reaction by which one ormore optically detectable labels are incorporated into the particles.

Substantial removal of the non-particle components of the stainingreaction means that at least about 50, 60, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99 or more percent of the non-particlecomponents of the staining reaction, i.e., substantially all componentsof the reaction other than the stained particles, are removed by one ormore physical separations, e.g., washing or centrifugation. In themethods provided herein, after staining, the resulting stained samplescan be diluted by a factor sufficient to reduce the background signalsassociated with optically detectable labels that are not bound to orassociated with the particles, without physical separation or isolation.Without being bound by theory, the analysis of the stained particles bydilution of the staining reaction mix, without a physical separation orisolation, can provide improved efficiency by reducing the number ofsteps used to process the particles prior to analysis. In addition, theanalysis of microparticles or nanoparticles generally involves handlingsmall volumes of samples containing the particles and the repeatedwashing or centrifugation/ultracentrifugation of small volumes can leadto loss of a fraction of the particles, thereby reducing the accuracy ofanalysis. The dilution can be by a factor sufficient to minimizeinterference from the signal associated with free optically detectablelabel that is not associated with the particle, while maintainingenhanced signal from the optically detectable label that is bound to orotherwise associated with the particle. Thus, in embodiments of themethods provided herein, particle-associated label is analyzed in thepresence of free label, the free label generating a minimal backgroundsignal that does not interfere with the detection of signal generated bythe particle-associated optically detectable label. The dilution alsocan be by a factor whereby multiple particles are not detectedsimultaneously, i.e., there is minimal to no “coincidence.” For example,the dilution can be by a factor of anywhere from about 2-fold to about5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000,100,000-fold or higher, depending on the sample. In some embodiments, nodilution of the staining reaction solution may be needed. In anexemplary embodiment, when the sample is plasma, the dilution of asample stained with 500 nM di-8-ANEPPS and 50 nM molecularmarker-specific probe can be by a factor of about 1000.

The label-bound particles in the sample that is diluted, post-staining,as described above, can be detected, characterized (e.g., its molecularcomponents identified and/or its origin identified—e.g., if the particleis an EV, its cell/tissue of origin can be identified) and/orquantitated based on detection of the bound optically-detectable label.Any label that can be detected by optical means can be used for analysesof the particles. The sample can be analyzed by visual inspection or canbe illuminated by an instrument capable of detecting an opticallydetectable label associated with a particle. The illumination wavelengthcan be tailored to detection of a particular particle-associatedoptically detectable label, whether the label is a surface area probe orvolume probe, or a molecular marker-specific probe. The intensity of thesignal from the particle-associated optically detectable label can bemeasured by the instrument detecting such signal or by a separateinstrument capable of measuring the intensity of a signal from anoptically detectable label associated with a particle. Exemplary opticalelements for selecting and dispersing light can include, but are notlimited to, band pass filters, dichroic mirrors or optical gratings forfiltering or dispersing light onto a detector. Exemplary detectors caninclude, but are not limited to, a photomultiplier tube (PMT), anavalanche photodiode, an avalanche photodiode array, a silicon-PMT, ahybrid PMT, a photodiode array, a charged cathode device (CCD), anelectron multiplied CCD, a CMOS sensor detector, or any suitablephotodetector. The measured intensity can be used to characterize theparticles in the sample according to presence or absence of theparticle, type/identity of the particle, source/origin of the particle,number of molecules of molecular marker on the particle, size of theparticle or quantity of the particle.

Samples

Samples that contain particles for analysis according to the methodsprovided herein generally include particles in a liquid medium. Theparticles can be analyzed by detection, identification, characterizationaccording to the presence of one or more molecular markers associatedwith the particles, or characterization according to the size of theparticles. Any samples containing particles in a liquid can be analyzedaccording to the methods provided herein.

Any aqueous or organic liquid medium containing particles, where theparticles are not dissolved in the liquid medium, are contemplated foruse in the methods herein. The liquid medium can be a solution thatincludes solutes dissolved in the liquid medium, such as buffers. Insome embodiments, the samples include a suspension of particles, or acolloidal suspension of particles, in the liquid medium. Exemplaryliquid media containing particles that can be analyzed according to themethods provided herein include, but are not limited to, blood, milk,water, solutions containing particles such as membrane vesicles,lipoproteins, viruses, virus-like particles, apoptotic bodies, syntheticliposomes or extracellular vesicles, and biological fluids other thanblood such as plasma, serum, urine, saliva, seminal fluid, lavages(e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic),cervical fluid, cervicovaginal fluid, cerebrospinal fluid, vaginalfluid, breast fluid, breast milk, synovial fluid, semen, seminal fluid,sputum, cerebral spinal fluid, tears, mucus, interstitial fluid,follicular fluid, amniotic fluid, aqueous humor, vitreous humor,peritoneal fluid, ascites, sweat, lymphatic fluid, lung sputum orfractions or components thereof.

In some embodiments, the sample containing particles is a biologicalsample. In embodiments, the biological sample includes a biologicalfluid. The biological fluid in the biological sample can include, but isnot limited to, blood, plasma, serum, urine, saliva, seminal fluid,lavages (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear,arthroscopic), cervical fluid, cervicovaginal fluid, cerebrospinalfluid, vaginal fluid, breast fluid, breast milk, synovial fluid, semen,seminal fluid, sputum, cerebral spinal fluid, tears, mucus, interstitialfluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor,peritoneal fluid, ascites, sweat, lymphatic fluid, lung sputum orfractions or components thereof. In certain embodiments, the biologicalfluid is blood, plasma or serum. In some embodiments, the biologicalfluid is cerebrospinal fluid.

In certain embodiments, the biological sample is extracted from a cellor tissue sample of a subject, such as a biopsy sample (e.g., cancerbiopsy), or is extracted from normal or cancer cell samples or normal orcancer tissue samples where the cell or tissue samples can be derived,e.g., from the liver, lung, kidney, spleen, pancreas, colon, skin,bladder, eye, brain, esophagus, head, neck, ovary, testes, prostate, thelike or combination thereof. In some embodiments, the biological samplethat is extracted from a cell or tissues sample of a subject includes abiological fluid.

In some embodiments, the biological sample includes particles derivedfrom a cancer biopsy, a cancer cell or a cancer tissue. Cancer biopsysamples, cancer cell types or cancer tissue types from which particlescan be present in the biological sample include, but are not limited to,liver cells (e.g., hepatocytes), lung cells, spleen cells, pancreascells, colon cells, skin cells, bladder cells, eye cells, brain cells,esophagus cells, cells of the head, cells of the neck, cells of theovary, cells of the testes, prostate cells, placenta cells, epithelialcells, endothelial cells, adipocyte cells, kidney cells, heart cells,muscle cells, blood cells (e.g., white blood cells, platelets), the likeand combinations of the foregoing. In embodiments, the cancer is aglioblastoma. In certain embodiments, the cancer is ovarian, lung,bladder or prostrate cancer. In some embodiments, the biological samplethat includes particles derived from a cancer biopsy, a cancer cell or acancer tissue further includes a biological fluid. In embodiments, thebiological fluid is blood, plasma, serum, saliva, urine or cerebrospinalfluid. In some embodiments, the cancer is ovarian, lung, bladder orprostrate cancer and the biological fluid is saliva, urine or serum. Incertain embodiments, the cancer is brain cancer. In some embodiments,the cancer is brain cancer and the biological fluid is cerebrospinalfluid. In embodiments, the brain cancer is glioblastoma.

In some embodiments, a sample can be blood and sometimes a bloodfraction (e.g., plasma or serum). As used herein, the term “blood”encompasses whole blood or any fractions of blood, such as serum andplasma as conventionally defined, for example. Blood also contains buffycoats. Buffy coats sometimes are isolated by utilizing a ficollgradient. Buffy coats can comprise white blood cells (e.g., leukocytes,T-cells, B-cells, platelets, and the like) and samples extracted frombuffy coats can include particles, e.g., extracellular vesicles (EVs),derived from these cells. Blood plasma refers to the fraction of wholeblood resulting from centrifugation of blood treated withanticoagulants. Blood serum refers to the watery portion of fluidremaining after a blood sample has coagulated. Fluid or tissue samplesoften are collected in accordance with standard protocols hospitals orclinics generally follow. For blood, an appropriate amount of peripheralblood (e.g., between 3-40 milliliters) often is collected and can bestored according to standard procedures prior to or after preparation.In some embodiments, a sample obtained from a subject can containcellular elements or cellular remnants. In some embodiments, cancercells may be included in the sample. In embodiments, the sample isobtained from a human subject. In certain embodiments, the human subjectis a cancer patient and in some embodiments, the human subject does nothave cancer.

Particles

Any particles that can bind to or otherwise associate with an opticallydetectable label are contemplated for analysis according to the methodsprovided herein. The particles can occur in nature, or can be syntheticor artificially prepared. The particles described herein and elsewherein this application can be the particles of interest, i.e., theparticles that are desired to be analyzed by the methods, or they can beused as optical standard particles for improved accuracy of measurementof the optical intensity.

Inert Particles

In certain embodiments, the particles can be inert particles that canassociate with an optically detectable label or can be modified forassociation with an optically detectable label. Such particles caninclude metalloids, non-limiting examples of which include boron andsilicon, the like and combinations thereof. A particle sometimes caninclude, consist essentially of, or consist of, silica (e.g., silicondioxide (i.e., SiO₂)). A particle sometimes can include one or moremetals including, but not limited to, iron, gold, copper, silver,platinum, aluminum, titanium, tantalum, vanadium, the like, oxidesthereof and combinations thereof. A particle sometimes can include glass(e.g. controlled-pore glass (CPG)), nylon, Sephadex®, Sepharose®,cellulose, a magnetic material or a plastic material. A particlesometimes is a polymer or includes more than one polymer. Non-limitingexamples of polymers include polypropylene (PP), polyethylene (PE),polyamide, high-density polyethylene (HDPE), low-density polyethylene(LDPE), polyester, polyvinylidenedifluoride (PVDF), polyethyleneteraphthalate (PET), polyvinyl chloride (PVC), polytetrafluoroethylene(PTFE), polystyrene (PS), high-density polystyrene, acrylnitrilebutadiene styrene copolymers, crosslinked polysiloxanes, polyurethanes,(meth)acrylate-based polymers, cellulose and cellulose derivatives,polycarbonates, ABS, tetrafluoroethylene polymers, poly(2-hydroxy ethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate),poly(vinyl alcohol), poly(acrylic acid), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactides (PLA), polyglycolides (PGA),poly(lactide-co-glycolides) (PLGA), polyanhydrides, polyorthoesters,polycyanoacrylates, polycaprolactone, the like, copolymers thereof andcombinations of the foregoing.

The particles can be solid particles or particles that contain internalvoids. The particles can have a regular (e.g., spheroid, ovoid) orirregular shape (e.g., rough, jagged), and sometimes can benon-spherical (e.g., angular, multi-sided).

Membrane Vesicles

In embodiments of the methods provided herein, the particles includemembrane vesicles. A membrane vesicle, as used herein, refers to aparticle that includes fluid enclosed within a lipid-containing outershell. The enclosed fluid can include additional components, such asproteins and small molecules. A lipid molecule typically includes atleast one hydrophobic chain and at least one polar head. When exposed toan aqueous environment, lipids often will self assemble into structuresthat minimize the surface area exposed to a polar (e.g., aqueous)medium. Lipids sometimes assemble into structures having a single ormonolayer of lipid enclosing a non-aqueous environment, and lipidssometimes assemble into structures comprising a bilayer enclosing anaqueous environment. In a monolayer structure, the polar portion oflipids (e.g., the head of the molecule in the case of phospholipids andother lipids commonly found in cell substrates) often is orientedtowards the polar, aqueous environment, allowing the non-polar portionof the lipid to contact the non-polar environment.

A vesicle also can be a lipid bilayer configured as a spherical shellenclosing a small amount of water or aqueous solution and separating itfrom the water or aqueous solution outside the vesicle. Membranevesicles also can contain a fluid with, optionally, one or moremolecular components, enclosed within a lipid bilayer. Because of thefundamental similarity to a cell wall, vesicles have been used to studythe properties of lipid bilayers. Vesicles also are readilymanufactured. A sample of dehydrated lipid spontaneously forms vesicles,when exposed to water. Spontaneously formed vesicles can be unilamelar(single-walled) or multilamellar (many-walled) and are of a wide rangeof sizes from tens of nanometers to several micrometers. A lipid bilayertypically includes a sheet of lipids, generally two molecules thick,arranged so that the hydrophilic phosphate heads point towards ahydrophilic aqueous environment on either side of the bilayer and thehydrophobic tails point towards the hydrophobic core of the bilayer.This arrangement results in two “leaflets” that are each a singlemolecular layer. Lipids self-assemble into a bilayer structure due tothe hydrophobic effect and are held together by non-covalent forces thatdo not involve formation of chemical bonds between individual molecules.

In some embodiments, lipid bilayers are natural, and in certainembodiments lipid bilayers are artificially generated. Natural bilayersoften are made mostly of phospholipids, which have a hydrophilic headand two hydrophobic tails (e.g., lipid tails), and form a two-layeredsheet as noted above, when exposed to water or an aqueous environment.The center of this bilayer contains almost no water and also excludesmolecules like sugars or salts that dissolve in water, but not in oil.Lipid tails also can affect lipid composition properties, by determiningthe phase of the bilayers, for example. A bilayer sometimes adopts asolid gel phase state at lower temperatures and undergoes a phasetransition to a fluid state at higher temperatures. Artificial bilayersof membrane vesicles can be any bilayers assembled through artificialmeans, as opposed to bilayers that occur naturally (e.g., cell walls,lipid bilayers that cover various sub-cellular structures).

The presence of certain lipids or proteins sometimes can alter thesurface chemistry of bilayers (e.g., viscosity or fluidity of lipidbilayers). Phospholipids with certain head groups can alter the surfacechemistry of a bilayer. Non-limiting examples of bilayer constituentsthat can alter the surface chemistry of bilayers include fats, lecithin,cholesterol, proteins, phospholipids (e.g., phosphatidic acid(phosphatidate), phosphatidylethanolamine (e.g., cephalin),phosphatidylcholine (e.g., lecithin), phosphatidylserine, andphosphoinositides such as phosphatidylinositol (PI),phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate(PIP2) and phosphatidylinositol triphosphate (PIP3),phosphatidylglycerol, ceramide phosphorylcholine, ceramidephosphorylethanolamine, ceramide phosphorylglycerol),phosphosphingolipids, glycolipids including gangliosides, surfactants,the like and combinations thereof.

Different types or forms of lipid compositions (e.g., monolayers and/orbilayers) can be found naturally or generated artificially. Non-limitingexamples of lipid compositions include monolayers (e.g., micelles),supported lipid bilayers, linear lipid bilayers and the like.

A protein, glycoprotein, glycolipid, nucleic acid or carbohydrate oftenis inserted into a structure (e.g., monolayer and/or bilayer) formed bythe lipid or amphiphilic material composition, or is encapsulated withinthe interior of the structure (membrane vesicle or other particle asdescribed herein). A protein that is inserted into the structure can bewater soluble, detergent-solubilized or incorporated into a lipidbilayer (e.g., vesicle, liposome) or a lipid monolayer (e.g., micelle)in some embodiments.

Some types of membrane vesicles can include lipoproteins, endosomes,apoptotic bodies, viruses, virus particles and virus-like particles.Endosomes are membrane-bound vesicles, formed via a complex family ofprocesses collectively known as endocytosis, and found in the cytoplasmof virtually every animal cell. The basic mechanism of endocytosis isthe reverse of what occurs during exocytosis or cellular secretion orthe release of extracellular vesicles (EVs, e.g., ectosomes, exosomes)as it involves the invagination (folding inward) of a cell's plasmamembrane to surround macromolecules or other matter diffusing throughthe extracellular fluid. The encircled foreign materials are thenbrought into the cell, and following a pinching-off of the membrane(termed budding), are released to the cytoplasm of the cell in asac-like vesicle. The sizes of the endosomal vesicles can vary andgenerally are nanoparticles. Endosomes larger than 100 nanometers indiameter typically are referred to as vacuoles.

Viruses, virus particles and virus-like particles can include a lipidbilayer and, in embodiments, carry proteins on their surface, includingenvelope proteins, coat proteins and cellular membrane proteins. “Nakedviruses” generally lack surface proteins and can be modified to includesurface proteins (e.g., by insertion of the proteins into the outerlipid bilayer of the virus). Viruses include for example, but are notlimited to, retroviruses and DNA viruses. Virus particles can includethe fully or partially assembled capsid of a virus. A viral particle mayor may not contain nucleic acid. Virus particles generally include oneor more of or two or more of the following: genetic material made fromeither DNA or RNA; a protein coat that protects the genetic material;and in some embodiments an envelope of lipids that surrounds the proteincoat when they are outside a cell.

Lipoproteins are globular, micelle-like particles that include anon-polar core of acylglycerols and cholesteryl esters surrounded by anamphiphilic coating of protein, phospholipid and cholesterol.Lipoproteins have been classified into five broad categories on thebasis of their functional and physical properties: chylomicrons, whichtransport dietary lipids from intestine to tissues; very low densitylipoproteins (VLDL); intermediate density lipoproteins (IDL); lowdensity lipoproteins (LDL); all of which transport triacylglycerols andcholesterol from the liver to tissues; and high density lipoproteins(HDL), which transport endogenous cholesterol from tissues to the liver.Lipoprotein particles undergo continuous metabolic processing and canhave variable properties and compositions. Lipoprotein densities canincrease without decreasing particle diameter because the density oftheir outer coatings is less than that of the inner core. The proteincomponents of lipoproteins are known as apolipoproteins. At least nineapolipoproteins are distributed in significant amounts among the varioushuman Iipoproteins.

Apoptotic bodies are released during apoptosis (programmed cell death).When a cell undergoes apoptosis, the structure of the cell breaks down.The breakdown components are packaged into apoptotic bodies, which caninclude membrane bound “sacs” that contain nucleic acids, proteins andlipids. When the ability of neighboring cells and/or macrophages toclear these breakdown components is overwhelmed by high numbers ofapoptotic bodies (“excessive” apoptosis) or defects in clearing thebodies, apoptotic bodies are released into circulation and can bedetected in blood plasma or serum (Holdenrieder et al, 2001a;Holdenrieder et al, 2001b; Holdenrieder et al, 2001c; Lichtenstein etal, 2001). Above-average levels of apoptotic bodies in the bloodstreamhave been correlated, e.g., with the presence tumors and cancers. An“apoptotic body” can contain nucleic acids, proteins, lipids, but nonucleus, although it may contain fragmented nuclei. In general,apoptotic bodies are less than 10 microns in size, generally betweenabout 25 nm, 50nm, 75 nm or 100 nm to about 150 nm, 200 nm, 250, 300 nm,350 nm, 400 nm, 400 nm, 500 nm, 1 micron, 1.5 micron, 2 microns, 2.5microns, 3 microns, 3.5 microns, 4 microns, 4.5 microns or 5 microns insize.

Liposomes

A liposome is an artificially prepared vesicle that includes at leastone lipid bilayer and also can be made of naturally occurring orsynthetic lipids, including phospholipids.

Liposomes can include MLV (multilamellar vesicles), SUV (SmallUnilamellar Vesicles), LUV (Large Unilamellar Vesicles) and GUV (GiantUnilamellar Vesicles). Unilamellar vesicles generally contain a singlelipid bilayer, while multilamellar vesicles generally include more thanone lipid bilayer. As used herein, “multivesicular liposome” refers toman-made, microscopic lipid vesicles containing lipid membranesenclosing multiple concentric or non-concentric aqueous chambers.

Various types of lipids can be used to make liposomes, including neutrallipids and amphipathic lipids. Examples of neutral lipids includediglycerides, such as diolefin, dipalmitolein; propylene glycol esterssuch as mixed diesters of caprylic/capric acids on propylene glycol;triglycerides such as triolein, tripalmitolein, trilinolein, tricaprylinand trilaurin; vegetable oils, such as soybean oil; lard or beef fat;squalene; tocopherol; and combinations thereof. Examples of amphipathiclipids include those with net negative charge, zero net charge, and netpositive charge at pH 7.4. These include zwitterionic, acidic orcationic lipids. Such exemplary amphipathic lipids include, but are notlimited to, phosphatidylglycerol (PG), cardiolipin (CL),phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol,phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin,diacyl trimethylammonium propane (DITAP), DOPC orDC18:1PC=1,2-dioleoyl-sn-glycero-3-phosphocholine; DLPC orDC12:0PC=1,2-dilauroyl-sn-glycero-3-phosphocholine; DMPC orDC14:0PC=1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC orDC16:0PC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC orDC18:0PC=1,2-distearoyl-sn-glycero-3-phosphocholine; DAPC orDC20:0PC=1,2diarachidoyl-sn-glycero-3-phosphocholine; DBPC orDC22:0PC=1,2-dibehenoyl-sn-glycero-3-phosphocholine;DC14:1PC=1,2-dimyristoleoyl-sn-glycero-3- phosphocholine;DC16:1PC=1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine;DC20:1PC=1,2-dieicosenoyl-sn-glycero-3-phosphocholine;DC22:1PC=1,2-dierucoyl-sn-glycero-3-phosphocholine;DPPG=1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol;DOPG=1,2-dioleoyl-sn-glycero-3-phosphoglycerol and combinations thereof.Additionally, lipoproteins, gangliosides, cholesterol or plant sterolscan be used to make, or are a part of, liposomes.

Some examples of lipid-polymer conjugates and liposomes are disclosed inU.S. Pat. No. 5,631.018, which is incorporated herein by reference inits entirety. Examples of processes to make multilamellar andunilamellar liposomes are known in the art (see e.g. U.S. Pat. Nos.4,522,803, 4,310,506, 4,235,871, 4,224,179, 4,078,052, 4,394,372,4,308,166, 4,485,054 and 4,508,703).

Extracellular Vesicles

In embodiments of the methods provided herein, the particles can beextracellular vesicles (EVs). The term “extracellular vesicles,” as usedherein, can include membrane vesicles secreted from cell surfaces(ectosomes), internal stores (exosomes), cancer cells (oncosomes), orreleased as a result of apoptosis and cell death. In addition to lipidmembranes, depending on their cell or tissue of origin, EVs can includeadditional components such as lipoproteins, proteins, nucleic acids,phospholipids, amphipathic lipids, gangliosides and other particlescontained within the lipid membrane or encapsulated by the EVs.

All cells likely release EVs, making them attractive clinical diagnosticand therapeutic targets for a range of diseases. Non-limiting examplesof normal or cancer cell types that can release EVs include liver cells(e.g., hepatocytes), lung cells, spleen cells, pancreas cells, coloncells, skin cells, bladder cells, eye cells, brain cells, esophaguscells, cells of the head, cells of the neck, cells of the ovary, cellsof the testes, prostate cells, placenta cells, epithelial cells,endothelial cells, adipocyte cells, kidney cells, heart cells, musclecells, blood cells (e.g., white blood cells, platelets), the like andcombinations of the foregoing. Because EVs are involved in cell-cellcommunication, their characterization casts light upon their role innormal physiology and pathology. EVs in biological fluids fluidsincluding saliva, urine and sera are being interrogated as biomarkers ofovarian, lung, bladder and prostate cancers.

Glioblastoma is the most common form of primary brain cancer and is oneof the deadliest of human cancers. Glioblastoma cells releaseextracellular vesicles (EVs) containing amplified and mutated geneticmaterials derived from the tumor. The circulating EVs significantlyexceed tumor-derived circulating tumor cells and tumor derivedcirculating DNA and RNA. The released EVs appear in the localenvironment, the sera and cerebrospinal fluid (CSF). Amplification ofEGFR is the most frequent genetic abnormality associated with GBM, andEGFR overexpression has been shown in up to 40% of cases. GBM also oftenexpresses EGFRvIII, a genomic deletion variant of EGFR that isconstitutively active and oncogenic. Thus, the analyses of GBM EVs offera potential tool for monitoring tumor presence, phenotypic/genotypicfeatures, and pathophysiology.

EVs are abundant in various biological fluids, including blood, urine,and cerebrospinal fluid, but because they are released by differentmechanisms and by many different cells types, EVs in biofluids can beheterogeneous. While multispectral optical methods can detect vesiclesthat have different molecular markers, the small average size of EVs canresult in small optical signals from labels bound to or otherwiseassociated with these small particles, making it a challenge to analyzethe EVs by optical methods.

EVs can be released by all normal and cancer cells. With a mean diameterof ˜100-200 nm, however, individual EVs have ˜1/10,000 the surface areaand ˜1/1,000,000 the volume of a whole cell, making them difficult todetect using available single cell analysis tools, includingconventional flow cytometry. As a result, most proteomic and genomicanalysis is performed in bulk on thousands or millions of EVs. However,EVs in biofluids come from many different cell types, and from differentlocations from within the cell (exosomes secreted from intracellularmulti-vesicular bodies, ectosomes/microvesicles shed from the plasmamembrane surface, membrane fragments released as a result of cellapoptosis, necrosis, etc). Thus, in a bulk analysis, the signature fromtumor EVs may be lost in the background of vesicles from other sources.Single EV measurement approaches such as nanoparticle tracking analysis(NTA) and resistive pulse sensing (RPS) can report particleconcentrations, but provide no information on the cell of origin. Thus,provided herein are methods of analyzing EV particles wherein the EVparticles are characterized for their size, quantity, number ofassociated specific molecular markers and/or cell/tissue of origin.

The size of the particles (e.g., inert particles, liposomes or EVs)analyzed according to the methods provided herein can include particlesin the size range (average length, width or diameter) of about or at 10nm to about or at 5 microns, but generally are in the range of about orat 50 nm to about or at 100, 125, 150, 175, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nm or 1.0 to1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 microns.

Optically Detectable Labels and Detection Methods

Optically Detectable Labels

Any method that can detect and analyze optically detectable labels canbe used in the methods provided herein. Exemplary optically detectablelabels can include, for example, chromophores, chemiluminescentmoieties, bioluminescent moieties, fluorescent moieties and metals. Suchlabels can be detected, for example, by visual inspection, byspectroscopy, by fluorescence spectroscopy, by fluorescence imaging(e.g., using a fluorescent microscope or fluorescence stereomicroscope),by flow cytometry and the like.

Exemplary chromophores include, but are not limited to,3,3′-diaminobenzidine (DAB); 3-amino-9-ethyl carbazole (AEC); Fast Red;FD&C Yellow 5 (Tartrazine); Malachite Green Carbinol hydrochloride;Crocein Scarlet 7B (Dark Red); Erloglaucine (Dark Blue); Crystal Violet(Dark Purple); Bromophenol Blue; Cobalt(II) Chloride Hexahydrate (Red);Basic Violet 3; Acid Blue 9; Acid Red 71; FD&C Blue 1 (Brilliant BlueFCF); FD&C Red 3 (Erythrozine); and FD&C Red 40 (Allura Red AC).Exemplary fluorophores include, but are not limited to, di-8-ANEPPS,di-4-ANEPPS, a carbocyanine dye (e.g., DiO, DiL), a PKH dye (exemplaryof which are PKH-26 and PKH-67), Dylight488, Brilliant Violet, PacificBlue, Chrome Orange, Brilliant Blue 515, phycoerythrin (PE), rhodamine,fluorescein, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 andAPC-Alexa750, Oregon Green®, derivatives of rhodamine (e.g., Texas Redand tetrarhodimine isothiocynate (TRITC)), AMCA, Alexa Fluor®, Li-COR®,CyDyes® or DyLight® Fluors); tdTomato, mCherry, mPlum, Neptune, TagRFP,mKate2, TurboRFP and TurboFP635 (Katushka). The fluorescent reagent canbe chosen based on desired excitation and emission spectra. Alsoexemplary of fluorescent reagents are macromolecules that emit anoptically detectable signal, including fluorescent proteins, such as agreen fluorescent protein (GFP) or a red fluorescent protein (RFP). Avariety of DNA sequences encoding proteins that can emit a detectablesignal or that can catalyze a detectable reaction, such as luminescentor fluorescent proteins, are known and can be used in the methodsprovided herein. Exemplary genes encoding light-emitting proteinsinclude, for example, genes from bacterial luciferase from Vibrioharveyi (Belas et al., (1982) Science 218:791-793), bacterial luciferasefrom Vibrio fischerii (Foran and Brown, (1988) Nucleic acids Res.16:177), firefly luciferase (de Wet et al., (1987) Mol. Cell. Biol.7:725-737), aequorin from Aequorea victoria (Prasher et al., (1987)Biochem. 26:1326-1332), Renilla luciferase from Renilla renformis(Lorenz et al, (1991) Proc Natl Acad Sci USA 88:4438-4442) and greenfluorescent protein from Aequorea victoria (Prasher et al., (1987) Gene111:229-233). The luxA and luxB genes of bacterial luciferase can befused to produce the fusion gene (Fab2), which can be expressed toproduce a fully functional luciferase protein (Escher et al., (1989)PNAS 86:6528-6532).

In embodiments, the optically detectable label can be conjugated to amolecule (e.g., a protein, an antibody, a lectin, a peptide, a nucleicacid, a carbohydrate, a glycan and the like) that binds to or otherwiseassociates with a molecular marker on the particle, or associateswith/intercalates into the particle membrane (e.g., when the particle isa vesicle, liposome or EV).

In some embodiments, the particles can be analyzed by Raman flowcytometry using Surface Enhanced Raman Scattering (SERS) from metalnanoparticles (Nolan et al., Methods, 57:272-279 (2012).

Flow Cytometry

In embodiments of the methods provided herein, the particles areanalyzed by flow cytometry. Any conventional flow cytometer, including aspectral flow cytometer, a hyperspectral flow cytometer, or an imagingflow cytometer can be used in the methods provided herein. In certainembodiments, the flow cytometry analysis is fluorescence-based ratherthan light scatter-based. Without being bound by theory, it is believedthat when the particles are nanoparticles, e.g., of a size that is lessthan 1 micron or about 900 nm, 800 nm, 700 nm, 650 nm, 600 nm, 550 nm,500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 190 nm, 180 nm,170 nm, 160 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm,85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm or less indiameter, light scatter based detection is not as sensitive due to thesmall size of the particles. In addition, more than one particle can bedetected simultaneously (“coincidence”), leading to errors indetermining particle size and/or count (concentration).

Fluorescence-based flow cytometry can be useful, for example, in theanalysis of nanoparticles. For example, extracellular vesicles (EVs)often are in a size range of between about 100-200 nm, or about 500 nmor less. The analysis of such nanoparticles by fluorescence based flowcytometry, according to the methods provided herein, can quantitativelydetermine the size of the nanoparticles by stoichiometric staining ofthe surface area using a fluorescent surface area probe or volume probe,whereby the fluorescent intensity is proportional to the surface area orvolume, respectively. In addition, fluorescence-based flow cytometrypermits the sensitive detection of the number and type of molecularmarkers, such as surface antigens, on the nanoparticles, such as EVs,thereby providing information regarding the tissues/cells from whichthey originate, as well as whether the tissues/cells are cancerous(based on the number of EVs, number of molecules of molecular markerand/or type of molecular markers detected on the EVs).

Exemplary flow cytometers that can be used in the methods providedherein, in addition to any conventional flow cytometer, can include flowcytometers that employ slow flow or signal integration times to allowsufficient time to register the fluorescence intensities ofnanoparticles, which are dimmer than microparticles. In someembodiments, the signal integration time of the flow cytometer isbetween about 0.5 μsec-5000 μsec; about 1 μsec-4000 μsec; about 5μsec-3000 μsec; about 10 μsec-2000 μsec; about 10 μsec-1000 μsec; about15 μsec-500 μsec; about 15 μsec-100 μsec; or about 20 μsec-50 μsec. Inembodiments, the flow cytometer can have a high laser power and highnumerical aperture collection optics for improved sensitivity. Forexample, in embodiments, the flow cytometer can have a fluorescenceresolution (R) of about 10 molecules FITC to about 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 molecules ofFITC, as measured in units of mean equivalent soluble fluorochromes(MESF).

In particular embodiments, the flow cytometer used in the methodsprovided herein can include instruments such as those described, forexample, in Zhu et al., ACS Nano 8:10998-11006 (2014) and Zhang et al.,Analytical Chemistry, 84:6421-6428 (2012). Exemplary commercial flowcytometers for use in the methods provided herein include, for example,FACSCalibur (BD Biosciences) and CytoFlex (Beckman Coulter/DanaherCorporation). In embodiments, the sample flow rate setting of the flowcytometer can be the “Low” setting as designated in the instrument. Incertain embodiments, the sample flow rate setting can be “Medium” or“High,” as designated in the flow cytometer instrument. An exemplaryimaging flow cytometer for use in the methods provided herein is theImageStream imaging flow cytometer from Amnis.

Detection and Analysis

In particular embodiments of the methods provided herein, a samplecontaining particles that are membrane vesicles (e.g., liposomes,extracellular vesicles) can be adjusted to a concentration suitable foroptimal staining with the optically detectable label (surface areaprobe, such as di-8-ANEPPS, or molecular marker-specific probe, such asfluorescently labeled annexin V, cholera toxin B subunit or anti-CD61).For example, for a fixed concentration of the optically detectablelabel, the sample can be serially diluted whereby a final particleconcentration of between about 1-5×10⁸ to about 1-5×10¹⁰ particles/μL(an average of about 1-5×10⁹ particles/μL) is obtained. The serialdilution of the sample can be from about 2-fold to about 100,000-fold ormore, depending on the sample. For example, when the sample is plasma,the dilution to reach a desired staining optimum is high, about 100-foldto about 10,000-fold, generally about 200-fold, due to the presence ofinterfering proteins in the plasma that bind to the label (e.g., thefluorescent label di-8-ANEPPS). When the sample is cerebrospinal fluid(CSF), urine or saliva, the dilution factor is less, about 5-fold toabout 100-fold, generally about 20-fold, due to lower amounts ofinterfering protein in these samples.

The samples are diluted in an isotonic buffer, such as PBS or HanksBalanced Salt Solution (HBSS) and a small amount of surfactant (betweenabout 0.005% to about 0.1%, in some embodiments about 0.01%) is added tofacilitate incorporation of the surface area probe into the particle. Inembodiments, the surfactant is a poloxamer and in some embodiments, thepoloxamer is Pluronic-127.

In embodiments of the method, a surface area probe or volume probe isused to analyze the particles. The surface area probe or volume probe isadded at a concentration, generally between about 1 nM to 1 μM, thatachieves stoichiometric staining of the particle surface area or volume,respectively, and provides a measurable optical signal that isproportional to the surface area or volume, respectively. For example,when the surface area probe is di-8-ANEPPS, the concentration of addedprobe is about 500 nM.

In some embodiments of the method, a molecular marker-specific probe isused to analyze the particles. The molecular marker-specific probe canbe any ligand that can bind to or otherwise associate with a molecularmarker on the particle and includes an optically detectable label.Ligands for exemplary molecular marker-specific probes can include, butare not limited to, proteins, antibodies, lectins, peptides, nucleicacids, carbohydrates or glycans that include an optically detectablelabel, such as a fluorophore, a fluorescent protein, a fluorescentpolymer, a tandem conjugate of a first fluorophore and a secondfluorophore, or an epitope, antigen or other moiety that can bind orotherwise associate with a second ligand that can be labeled with anoptically detectable label.

The molecular marker-specific probe is added at a concentration,generally between about 1 pM to 100 μM, to produce stoichiometricbinding or association with the molecular markers, whereby the opticalsignal is proportional to the number of molecular markers per particle.For example, when the molecular marker-specific probe is fluorescentlylabeled annexin V, the concentration of added probe is about 25-100 nM.

In embodiments of the method, more than one probe selected from asurface area probe, a volume probe or a molecular marker-specific probecan be added to the particles for analysis.

Staining of the particles with the probes is performed for an amount oftime and at a temperature suitable for effective labeling and can be atambient temperature (20-25° C.) or any suitable temperature above thephase transition of the particle being detected. The incubation time canbe from about 30 seconds to about 1 minute, 2 minutes, 5 minutes, 10minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours or longer, depending on the particles and the opticallydetectable labels. For example, when the sample is plasma and the labelsare di-8-ANEPPS (surface area probe) and fluorescently labeled annexin V(molecular marker-specific probe), the incubation is for 1 hour atambient temperature.

After staining, the sample is diluted to reduce background signals fromfree label and adjust the concentration of label-associated/boundparticles for optimal measurement and analysis of the signal from thebound/associated label. For example, a plasma sample stained with 500 nMdi-8-ANEPPS and 50 nm fluorescently labeled annexin V is diluted1000-fold, prior to detection of the labels.

The stained particles are illuminated at a wavelength suitable fordetection of the surface area/volume probe and/or the molecularmarker-specific probe that is bound to or otherwise associated with theparticles. In exemplary settings, when the particle analysis is by flowcytometry and the optically detectable labels are di-8-ANEPPS andfluorescently labeled annexin V, a 160 mW, 488 nm laser is used toilluminate the particles. Exemplary wavelengths can include any suitablewavelength for detecting an optically detectable label. Exemplarywavelengths for fluorescent surface area probes or volume probes are,e.g., 457 nm, 472 nm, 488 nm, 492 nm (di-8-ANEPPS) and for fluorescentmolecular marker-specific probes are, e.g., 365 nm, 375 nm, 405 nm, 457nm, 472 nm, 488 nm, 492 nm, 514 nm, 532 nm, 561 nm, 633 nm, 635-642 nm,660 nm, 785 nm, 800 nm, 1064 nm.

The signals from the particle-associated optically detectable labels canbe detected using any detector suitable for effective detection of thesignals. Exemplary optical elements for selecting and dispersing lightcan include, but are not limited to, band pass filters, dichroic mirrorsor optical gratings for filtering or dispersing light onto a detector.Exemplary detectors can include, but are not limited to, aphotomultiplier tube (PMT), an avalanche photodiode, an avalanchephotodiode array, a silicon-PMT, a hybrid PMT, a photodiode array, acharged cathode device (CCD), an electron multiplied CCD, a CMOS sensordetector, or any suitable photodetector. In exemplary settings for aflow cytometer, a 600 nm long pass filter and PMT can be used to detectdi-8-ANEPPS and a 525/30 band pass filter and PMT can be used to detectfluorescently labeled annexin V.

In embodiments, when the analysis is by flow cytometry, the signalintegration times are adjusted according to the brightness (signalintensity from the particle-associated optically detectable label) ofthe particles being analyzed and the type of flow cytometer used toperform the analysis. For example, when the particles are small, e.g.,nanoparticles, fewer numbers of fluorescent labels becomes associatedwith the particles, thereby producing dimmer particles. To maximizedetection of fluorescent intensity in the measurement volume, signalintegration times are extended for smaller particles. In someembodiments, the signal integration time of the flow cytometer isbetween about 0.5 μsec-5000 μsec; about 500 μsec-5000 μsec; about 1μsec-4000 μsec; about 5 μsec-3000 μsec; about 10 μsec-2000 μsec; about10 μsec-1000 μsec; about 15 μsec-500 μsec; about 15 μsec-100 μsec; about20 μsec-50 μsec; or about 10, 15, 20, 25, 30, 35, 40, 45 or 50 μsec. Inan exemplary embodiment, when the sample is plasma, the signalintegration time is about 20 μsec.

An “optical standard particle,” as used herein, is a particle that canbe used as a calibration standard in the optical methods providedherein, for determining characteristics such as the size (e.g.,diameter) and/or surface area/volume and/or quantity of particles forwhich these characteristics are heretofore unknown (and one or more ofthese characteristics are known for the optical standard particle). Forexample, an optical standard particle of known size, or a preparation ofoptical standard particles of a known distribution of sizes (the sizeshaving been determined by a method not involving use of the opticallydetectable label, e.g., NTA, TRPS, EM) can be used to calibrate opticalintensity in terms of surface area. For example, the diameters of theoptical standard particles can be determined using NTA, and theequivalent surface area distribution calculated from the diameters. Theoptical standard particles can then be labeled with a surface area probeor volume probe and the signal intensities of the probe associated withthe particles can be measured and plotted against the surface areas ofthe particles, thereby obtaining a correlation between optical intensityand surface area or volume, respectively, that can be used to determinethe surface area or volume, based on measured optical intensity, of aparticle of unknown size.

For calibrating optical intensity in terms of numbers of molecules ofmolecular markers on the particles, an optical standard particle havinga known number of molecular marker molecules can be used, or apreparation of optical standard particles having a distribution ofdifferent known numbers of molecular marker molecules associated withthe particles can be used. The numbers of molecular marker molecules onthe optical standard particles can be determined by labeling theparticles with molecular marker-specific probe, then calibrating theirintensity against an external standard. For example, if the opticalintensity is fluorescence intensity, the external standard can be thefluorophore FITC (fluorescein isothiocyanate) or PE (phycoerythrin) andthe intensity is expressed as mean equivalent soluble fluorochromes(MESF). A correlation between MESF values and the fluorescence intensityvalues of the molecular marker-specific probes associated with theoptical standard values can be obtained, and the number of probemolecules/particle determined from the corresponding MESF values. Themolecular marker concentration can be determined by dividing the valueof probe molecules/particle by the particle surface area.

An optical standard particle, as used herein, also can be alipid-containing particle including, but not limited to, a liposome, EVor other lipid-containing vesicle, containing a known amount of lipid.Such particles can be stained in one or more known amounts using one ormore known amounts of probe, thereby obtaining one or more stainingsolutions containing optical standard particles associated with probe atone or more known probe to lipid ratios. These staining solutionscontaining optical standard particles stained at known probe to lipidratios can then be used, for example, to obtain a correlation betweenthe values of the ratios and the detection of a spectral shift, asdetermined by a change in optical wavelength at which the maximumoptical intensity of the stained optical standard particles is detected,or as determined by the ratio of optical intensities at two opticalwavelengths. A probe to lipid ratio at which a spectral shift isdetected, or a change in ratio of optical intensities is determined, isidentified as a probe to lipid ratio that approaches or is atsaturation. The probe to lipid ratio of the optical standard particlethat is identified as a probe to lipid ratio that approaches or is atsaturation can be used to determine the amount of probe to be added tothe sample containing particles to be analyzed, whereby the resultingprobe to lipid ratio of the particles to be analyzed approaches or is atsaturation. The correlation can be predetermined, or the samplecontaining particles to be analyzed can be spiked with one or more knownamounts of the optical standard particle to determine the amount ofprobe that results in a probe to lipid ratio that approaches or is atsaturation.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology. Certain examples set forth below utilize standardrecombinant DNA, membrane vesicle/liposome preparation and otherbiotechnology protocols known in the art.

Example 1 Materials and Methods

This example describes the materials and procedures used in exemplaryembodiments of the methods as demonstrated herein.

1. Materials

All lipids, including: L-α-phosphatidylcholine (Egg, Chicken) [PC];L-α-phosphatidylethanolamine (Egg, Chicken) [PE]; GM1 ganglioside(Brain, Ovine-Ammonium Salt) [GM1]; L-α-phosphatidylserine (Brain,Porcine) (sodium salt) [PS]; Sphingomyelin (Brain, Porcine); Cholesterol(Ovine, wool >98%);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodiumsalt) [PE-b]; and 1-palmitoyl-2- (dipyrrometheneborondifluoride)undecanoyl-sn-glycero-3-phosphocholine [Top Fluor PC] werepurchased from Avanti Polar Lipids. Di-8-ANEPPS(4-[2-[6-(Dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium)was from Biotium; DiOC6(3) iodide (3,3′-Dihexyloxacarbocyanine iodide)and PKH67 were from Sigma. Nile Red fluorescent beads (0.53 μm and 0.11μm) were from Spherotech.

Recombinant Annexin V and hamster anti-ratCD61 (clone 2C9.G2, BioLegend)were labeled with Dylight488-succinimidyl ester (Pierce) and the F/Pratios determined following the manufacturer's instructions. PPAK(D-Phe-L-Pro-L-Arg-chloromethyl ketone) was from HaematologicTechnologies Inc. Phosphate buffered saline (PBS) without Ca⁺⁺ and Mg⁺⁺was from Corning. All other reagents were from Sigma-Aldrich. Glasstubes were from Fisher and microfuge tube were from Axygen. All buffers,diluents, and sheath fluid was filtered through a 0.1 um pore diameterfilter (Pall Corporation) before use.

2. Preparation of Liposomes

Lipids were dissolved in chloroform at concentrations varying between2.5-100 mg/ml. The composition (mole percent) of lipid vesicles was47.9% PC, 16% PE, 15% sphingomyelin, 13% cholesterol, 6% PS, 1% GM1, and1% PE-b. The lipids, prepared at the described molar ratio at a finalamount of 10 μmoles total lipid, were added to a 12×75 mm test tube. Thechloroform solvent was slowly evaporated in a fume hood under a lightstream of N₂ gas, until a thin lipid film remained around the bottom ofthe tube. The thin lipid film was then hydrated with 1 mL of 0.1 μmfiltered PBS (no Ca⁺⁺, Mg⁺⁺) buffer and vortexed vigorously. Theresulting multi-lamellar vesicles (MLVs) were subjected to threefreeze/thaw cycles alternating between an ethanol bath on dry ice and a40° C. warm water bath. Following the freeze/thaw cycles the solutionwas incubated for an additional 60 minutes, with vortexing every 15minutes. MLVs were aliquoted into 100 μL volumes and stored at −20° C.To prepare unilamellar vesicles, an aliquot of MLVs, prepared asdescribed above, was diluted ten-fold in 0.1 μm filtered PBS (no Ca++,Mg) to a total lipid concentration of 1 mM in preparation for extrusionto unilamellar liposomes. MLVs were extruded through a LipsoFastextrusion device (Avestin) fitted with Nucleopore polycarbonate filtermembrane with average pore diameters of 0.4 μm, 0.2 μm, 0.1 μm, 0.08 μm,and 0.05 μm (GE Water & Process Technology) for a total of 21 passes (anodd number so as to minimize contamination with un-extruded particles infinal syringe). Liposomes were collected in 1.5 mL microfuge tubes,aliquoted, and stored for up to several weeks at 4° C.

3. Cell-free Plasma Supernatants containing Extracellular Vesicles (EVs)

Female Sprague Dawley rats (11 weeks of age) were obtained from CharlesRiver Laboratories (Wilmington, Mass.) and cared for in accordance withthe Guide for the Care and Use of Laboratory Animals, 8th Edition (26).All animals were determined to be pathogen free by Charles RiverLaboratories assessment plus profile testing. Animals were individuallyhoused at an AAALAC, Intl-accredited facility in non-sterile ventilatedpolycarbonate micro-isolator cages on corncob bedding. All researchprotocols were approved by Amgen Inc. (Seattle, Wash.) InstitutionalAnimal Care and Use Committee. Animals had ad libitum access to pelletedfeed (2020× Teklad; Harlan Laboratories Inc., Madison, Wis.) and water(reverse osmosis-purified) via automatic watering system. Animals wereon a 12:12 hr light:dark cycle in rooms with controlled temperature andhumidity and had access to enrichment opportunities (enrichment tubesand Nylabones). Blood (˜400 μL) was collected into sodium citrate, andcentrifuged 10 minutes at 2500×g and the supernatant collected toprepare cell-free plasma which was aliquoted and stored at −80° C.

4. Nanoparticle Tracking Analysis (NTA)

Liposomes were diluted in filtered DPBS and loaded into the chamber of aNanosight LM-20 equipped with a 532 nm laser and a CCD (charged cathodedevice) camera. The optimal camera level (setting: 15) and threshold(setting: 2) were established in preliminary experiments. Five movies of60 seconds each were recorded and analyzed for each sample using theNanoSight software. Average histograms and mean diameters are reported.

5. Flow Cytometry Instrumentation and Operation

Samples were analyzed on a FACSCalibur (BD Biosciences) equipped withstock lasers, filters, and detectors and using the “Low” sample flowrate setting. Samples also were analyzed using a flow cytometer ofhigher sensitivity constructed using components from commercialinstruments. The constructed flow cytometer contained an optical bench(flow cell, excitation laser beam shaping optics, forward angleobscuration bar, orthogonal collection optics, and optical relay fibers)from a FACSCanto flow cytometer (BD Biosciences, San Jose, Calif.), a488 nm laser (200 mW, Sapphire, Coherent), and a multi-PMT detectorassembly from a Beckman Coulter Elite cell sorter. Green (DyLight 488)and red (di-8-ANEPPS) fluorescence was collected through a 525/40bandpass or a 600 LP, respectively. Sheath and sample flow was providedby continuous flow pumps (Milligat, GlobalFIA, Fox Island, Wash.) atrates of 20 μL/sec and 0.02 μL/sec respectively, which gave a transittime (pulse width) of ˜20 μsec through the probe volume (about 10×longer than a conventional flow cytometer). Data was acquired with acustom data acquisition system that digitized signal pulses from theanalogue detectors (PMTs and photodiodes) and recorded pulse height andarea from each channel, plus pulse width from the trigger channel. Somehistogram data was analyzed using the Kolmogorov-Smirnov (KS) test (27).

6. Vesicle Staining and Size Calibration

Samples (liposomes or cell-free plasma supernatants) were seriallydiluted into 100 μL of 0.1 μm filtered HEPES buffered saline (HBS; 150mM NaCl, 10 mM HEPES pH 7.4) containing 500 nM di-8-ANEPPS, 0.01%Pluronic-127, 5 mM CaCl₂ and 20 μM PPAK in a row of a 96 well plate,stained for at least 60 minutes, then diluted 1:800 in PBS and analyzedby flow cytometry to determine the dilution of sample that gave optimalstaining. For liposomes, this was at a probe to lipid ratio of at least0.1, which was equivalent to 5×10¹⁰ particles/mL (measured by NTA) in500 nM di-8-ANEPPS. For EVs in rat plasma, high concentrations of plasmaproteins that can also bind di-8-ANEPPS required the use of a higherprobe to lipid ratio, achieved at a concentration of 5×10⁹nanoparticles/mL (as measured by NTA) in 500 nM Di-8-ANEPPPS. Insubsequent experiments, EV preps were diluted in staining buffer asabove, 50 nM of DyLight488-labeled surface marker added, and the samplestained for at least 60 minutes (determined in preliminary experiments)at ambient temperature, followed by dilution and analysis by HSFC.

To calibrate the di-8-ANEPPS fluorescence in terms of vesicle surfacearea, the di-8 intensity (channel number) was plotted against surfacearea (calculated from NTA diameter estimates, assuming sphericity) atfrequency intervals of 0.1. The slope and intercept of this line wasused to convert channel number to surface area (nm²), and then diameterwas calculated with the assumption that the vesicles are spherical.

Example 2 Fluorescence Detection vs. Light Scatter based Detection

Conventional flow cytometry on particles≧1 μm generally use lightscatter as a trigger parameter because the scatter intensity of suchparticles generally is well above background. Scatter intensity howeverdrops off rapidly as particle size decreases and particle refractiveindex, collection angle, and “cleanliness” of sheath and sample bufferscan have large effects on the performance of this method. The resolutionby flow cytometry using fluorescence as a trigger was compared to theresolution obtained using light scatter as a trigger. Establishedfluorescence calibration protocols were used (Nolan J P, Stoner S A. Atrigger channel threshold artifact in nanoparticle analysis, CytometryPart A 2013;83A:301-305; Hoffman R A, Wood J C S. Characterization ofFlow Cytometer Instrument Sensitivity, Current Protocols in Cytometry:John Wiley & Sons, Inc.; 2007; Schwartz A, Gaigalas A K, Wang L, Marti GE, Vogt R F, Fernandez-Repollet E. Formalization of the MESF unit offluorescence intensity, Cytometry Part B: Clinical Cytometry2004;57:1-6; Chase E S, Hoffman R A. Resolution of dimly fluorescentparticles: A practical measure of fluorescence sensitivity, Cytometry1998;33:267-279; Wood J. Fundamental flow cytometer properties governingsensitivity and resolution, Cytometry 1998;33:260-266; Wood J, Hoffman RA. Evaluating fluorescence sensitivity on flow cytometers: an overview,Cytometry 1998;33:256-259; Schwartz A, Fernandez Repollet E, Vogt R,Gratama J W. Standardizing flow cytometry: construction of astandardized fluorescence calibration plot using matching spectralcalibrators, Cytometry 1996;26:22-31; Schwartz A, Marti G, Poon R,Gratama J, Fernández-Repollet E. Standardizing flow cytometry: aclassification system of fluorescence standards used for flow cytometry,Cytometry 1998;33:106-114; Wang L, Gaigalas A K, Abbasi F, Marti G E,Vogt R F, Schwartz A. Quantitating fluorescence intensity fromfluorophores: practical use of MESF values. JOURNAL OF RESEARCH-NATIONALINSTITUTE OF STANDARDS AND TECHNOLOGY 2002;107:339-354).

A mixture of two Nile Red fluorescent beads, 0.53 μm and 0.11 μm, wereanalyzed by flow cytometry using either light scatter-based triggeringor fluorescence-based triggering, at a concentration adjusted tominimize coincidence (simultaneous detection of more than one particlein the measurement volume, as a single count) to a negligible level(˜1×10⁷ particles/mL). FIG. 1 shows side scatter and fluorescenceintensity histograms of the particle measurements triggered onfluorescence (A-C) vs. scatter (D-F). The trigger threshold required tominimize the background event rate threshold (dashed line) and thesystem background signal levels obtained using a software trigger (greyfilled histogram) also are shown.

The histograms of FIG. 1 show that the 0.53 μm bead is readily detectedusing a side scatter trigger or a fluorescence trigger. Single beads areclearly identified on both side scatter and Nile Red fluorescencechannels, as are coincident events, which are approximately twice asbright. However, the light scatter from the 0.11 μm bead is much lowerand as the trigger threshold is lowered, triggered events from particlesin the sheath and sample fluids begin to dominate the histogram, evenbefore the optical background of the system as determined by a softwaretrigger is reached. By contrast, setting the fluorescence triggerthreshold just above the software trigger background allows the dim(0.11 μm) fluorescent beads to be detected, while their light scatter isin background range. The population of 0.11 μm beads could be resolvedabout background in the fluorescence channel (see, e.g., panels B andC), but in the side scatter channel were poorly resolved from noise andbelow the threshold required for side scatter triggering (see, e.g.,panels E and F)

The results show that in this system, with sheath and sample fluidconsisting of 0.1 μm filtered nanopure water, fluorescence triggeringprovides superior resolution. Thus, fluorescence triggering was expectedto be even more significant as a tool for the study of lipid membranevesicles, whose refractive index is significantly lower than thepolystyrene Nile Red beads studied in this example and, as aconsequence, are expected to scatter less light.

Example 3 Vesicle Staining using Surface Area Probes

Several lipophilic fluorescent probes were evaluated for their abilityto stain membrane vesicles as surface area probes. Surface area probeswere assessed for their ability to bind saturably and stoichiometricallywith membrane vesicles such that probe-associated fluorescence isproportional to surface area and the fluorescence is enhancement whenbound to membrane relative to when the probe is free in solution.

Three blue-excited membrane surface area probes, DiO, PKH67, anddi-8-ANEPPS, were evaluated for their suitability in staining lipidvesicles for analysis by flow cytometry. Absorbance and fluorescenceemission spectra of each probe were obtained in PBS and in PBScontaining 100 nm diameter liposomes. All three probes exhibited anincrease in fluorescence upon addition of lipid membrane, withDi-8-ANEPSS having the lowest fluorescence in aqueous buffer and thegreatest enhancement in the presence of lipid membrane.

The suitability of these probes for detecting liposomes was evaluatedusing flow cytometry. Different dilutions of a liposome stock solutionwere stained using a fixed concentration of probe (100 nM) to givedifferent probe/lipid ratios (P/L) and measured using the custom flowcytometer set for triggering on fluorescence. For all probes, both thenumber of events per uL of liposome stock solution and the eventbrightness varied for P/L ratios>0.1. However, for di-8-ANEPPS, both theliposome concentration estimates and the liposome brightness wereconsistent for P/L>0.1 and up to a P/L of 0.25, indicating that thedi-8-ANEPPS staining intensity of liposomes is saturable over this P/Lrange.

To confirm that single vesicles and not coincident vesicles were beingmeasured (coincidence often is a problem when attempting to measure verydim small particles), a series of two fold dilutions ofdi-8-ANEPPS-stained liposomes was performed, and it was found that themeasured event rate decreased in proportion to the dilution, but themedian fluorescence did not change, consistent with the analysis ofsingle particles. The liposome concentration measured by flow cytometry,after accounting for dilutions (6.2×10⁸ particles/uL), was within afactor of two of the concentration estimated by NTA (12×10⁸particles/uL). To confirm that lipid vesicles were being measured, thedi-8-ANEPPS-stained liposomes were treated with with 0.05% Triton X-100,which solubilized the vesicles and reduced particle counts to backgroundlevels.

The extruded liposomes were found to be stable, with stored liposomes (4months, 4° C.) retaining the same fluorescence staining properties asfreshly extruded liposomes. The results demonstrate that liposomes areattractive as possible reference particles to aid in the standardizationof EV measurements.

Example 4 Calibration of Vesicle Size using NTA or Flow Cytometry

The results from Example 3 above suggested that di-8-ANEPPS intercalatesinto membrane vesicles in a stoichiometric manner, and thus could beuseful to estimate vesicle surface area and, therefore, vesiclediameter. To evaluate this possibility, di-8-ANEPPS at a P/L ratioof>0.1 was used to measure liposomes that had been prepared by extrusionthrough nucleopore filters with different pore sizes. FIGS. 2A-D depictfluorescence intensity histograms of di-8-ANEPPS stained vesiclesprepared by extrusion through polycarbonate membrane filters withaverage pore sizes of 200 nm (A), 100 nm (B), 80 nm (C) and 50 nm (D),as described in Example 1. FIGS. 2E-H depict nanoparticle diameterpopulation histograms from NTA of the same vesicles, with the mediandiameter of each preparation indicated.

As shown in FIGS. 2A-D, liposomes extruded through increasingly smallerpore sizes and stained with di-8-ANEPPS showed unimodal fluorescenceintensity distributions with increasingly lower intensities. Forreference, these intensities ranged from ˜25-1000 mean equivalentPE-TexasRed molecules (MEPETR), determined using Spherotech Rainbowbeads and the manufacturer's calibration values. These same liposomepreparations also were analyzed by nanoparticle tracking analysis (NTA),which estimates nanoparticle diameter from its Brownian diffusion andfrom which diameter can be calculated (FIGS. 2E-H, dashed lines).

To calibrate fluorescence intensity in terms of surface area, the NTAdiameter measurements were converted to surface area using theassumption of sphericity, and the resulting distribution plotted againstthe di-8-ANEPPS intensity distribution at frequency intervals of 0.1(FIG. 3). The slope and intercept of the line were used to convertdi-8-ANEPPS intensity into surface area units, and then to diameterunits (FIGS. 2E-H, solid lines). For vesicles ˜120 nm and smaller, therewas excellent concordance between the fluorescence intensitymeasurements and NTA. For larger vesicles (larger than ˜200 nm), flowcytometry using a fluorogenic membrane surface area probe was found todetect the vesicles more efficiently when compared to NTA.

The light scatter intensity of the events detected using fluorescencetriggering also was recorded; it was found that light scatter from lowrefractive index membrane vesicles was below the detection limit of theflow cytometer instrument. Light scatter measurements also are sensitiveto the presence of non-fluorescent particles in the sample and sheathfluids and even when particles can be detected, calibration of thesemeasurements is difficult. Thus, the results demonstrate thatfluorescence staining of vesicle membrane surface area is an attractivealternative to light scatter-based approaches for detecting andestimating the size of very small vesicles by flow cytometry.

Example 5 Analysis of Extracellular Vesicles in Plasma

To evaluate flow cytometry for EV detection, plasma from untreated ratswas analyzed using the custom flow cytometer described in Example 1.Cell-free plasma was prepared from blood by low speed centrifugation (2×2500×g, 10 minutes) to remove large cells and analyzed using NTA todetermine total nanoparticle concentration and population sizedistribution. Plasma from three different rats was measured by NTA(FIGS. 4A-C) or by flow cytometry (vesicle flow cytometry or VFC; FIGS.4D-F).

For flow cytometry, samples were diluted to approximately 1×10⁹nanoparticles per uL, stained with 100 nM di-8-ANEPPS, further diluted1000-fold, and analyzed by flow cytometry. FIG. 4 shows NTA (4A-C) andflow cytometry (4D-F) population diameter distributions from the threerepresentative rat plasma samples, estimated using liposomes as areference particle as described above. Both NTA and flow cytometryreported unimodal particle populations, with a peak (mode) diameter of˜120 nm.

The correlation between NTA nanoparticle concentration estimates andflow cytometry vesicle concentration estimates for eight differentanimal samples are presented in

FIG. 4G. NTA reported total nanoparticle concentrations in untreated ratplasma between 1.54×10⁷/uL and 1.24×10⁹/uL (average 4.87×10⁸/uL, n=8),while flow cytometry reported total vesicle concentrations of5.12×10⁷/uL to 1.95×10⁸/uL (average 1.03×10⁸/uL, n=8). In general adirect correlation was found between NTA, which measures particles withdetectable light scatter, and flow cytometry, which measures particleswith detectable fluorescence, with NTA reporting concentrations ˜3-5fold higher than flow cytometry. This difference may be due in part tothe selectivity of flow cytometry for membranous particles, as well asuncertainties in the volume of the sample measured by NTA and the limitsof detection of the two methods.

To test the ability of flow cytometry to measure high abundance surfacemarkers on EVs, platelet rich plasma (PRP) from rats were prepared andstimulated with the ionophore A23187, which is known to induce plateletEV release. After treatment, samples were centrifuged as above to removeresidual cells and platelets and stained with di-8-ANEPPS as a surfacearea probe, followed by staining with either Dylight488-Annexin V or-CD61 as a membrane molecular marker-specific probe. CD61 is a componentof the α2β3 integrin complex found in high abundance on platelets, whileannexin V binds to exposed phosphatidylserine (PS) on the outer leafletof the vesicle membrane.

FIG. 5 depicts the fluorescence measurement of EV surface markers fromcontrol plasma (5A, 5B) or ionophore-treated platelet-rich plasma (5C,5D). As shown in FIG. 5, EVs stained with both di-8-ANEPPS and Dylight488-Annexin V show a clearly resolved population of EVs, with annexin Vbinding corresponding to ˜28% of total EVs with a median brightnessequivalent to ˜6400 MESF of fluorescein, as well as a population thatwas annexin V negative (FIG. 5A). Upon treatment with ionophore, >60% ofthe EVs were annexin V positive (FIG. 5C). Similarly, ˜23% of EVs incontrol plasma were positive for CD61, with a median brightnessequivalent to ˜1500 MESF of fluorescein (FIG. 5B), which increased to˜50% CD61 positive in ionophore treated PRP (FIG. 5D). Thus, highsensitivity flow cytometry triggered on the fluorescence of afluorogenic membrane probe allows detection of individual EVs in plasma,as well as the detection of EV sub-populations expressing cell surfacemarkers.

Example 6 Analysis of Extracellular Vesicles in Cerebrospinal Fluid

Cerebrospinal fluid (CSF) samples from patients diagnosed with fivedifferent neurological disorders, and from normal subjects, wereanalyzed by NTA or by flow cytometry. For flow cytometry, CSF sampleswere stained using di-8-ANEPPS as a fluorescent surface area probe forthe membranes of EVs in CSF, and DyLight488-labeled surface markers(annexin V or anti-CD41) were used as fluorescent molecularmarker-specific probes of the EVs. The samples prepared for flowcytometry were analyzed using the custom constructed instrumentdescribed in Example 1.

Pools of patient-derived CSF samples from patients having high gradeglioma, low grade glioma, Alzheimer's disease, Parkinson's disease orsubarachnoid haemorrhage showed 2- to 7-fold increases in theconcentration of EVs, compared to samples from normal subjects, with nonotable differences in size, indicating that the EV concentrations couldhave diagnostic value.

Further, while the EV size distributions determined by NTA and by flowcytometry (measuring fluorescence intensity of EV-associateddi-8-ANEPPS) were similar, the particle concentrations obtained by flowcytometry were lower than NTA (2.16×10⁶ particles/μl with flowcytometry, relative to 4.32×10⁶ particles/μl with NTA) and the mean sizewas larger (192 nm with flow cytometry, relative to 118 nm with NTA),indicating that NTA likely measures all scattering particles in the CSFwhile flow cytometry measures membranous nanoparticles. In addition, themeasured intensities of the fluorescent molecular marker-specific probesassociated with the EVs revealed variable annexin V—associated EVfractions, with some showing a significant fraction of CD41—associatedEVs. Thus, fluorescence measurement based flow cytometry couldcharacterize the EVs in CSF with high sensitivity compared to NTA, withflow cytometry providing fluorescent antibody based speciation of EVs.

Example 7 Antibody-Capture Nanospheres for Compensation and SpectralReference

A set of 0.45 um antibody capture beads that each capture about 5000antibody molecules were developed as positive control nanobeads (opticalstandard particles) and BSA coated beads were developed as negativecontrol nanobeads (optical standard particles). 450 nm polystyrene beadswere coated with either anti-lambda IgG (positive control) or BSA(negative control). Such beads can serve as a spectral reference for usein compensation or spectral unmixing, to obtain a more accuratedetection and/or quantitation of the fluorescence intensity ofnanoparticles (e.g., EVs) of interest. FIG. 6 depicts histograms from asample containing both the positive and negative control beads stainedwith a DyLight488-conjugated antibody. The results show that theantibody capture beads were selectively stained by the fluorescentantibody relative to the BSA beads, as measured by DyLight488fluorescence, thereby demonstrating that these spectral reference beadscan be added to a nanoparticle sample in known amounts to quantitate therelative numbers of molecular marker molecules associated with thenanoparticles (i.e., the molecules associated with the nanoparticlesthat bind to fluorescent molecular marker-specific probes).

The antibody capture beads can be used as compensation standards forflow cytometry measurements of particle, including nanoparticle, surfacemarkers or in multispectral measurements of particle, includingnanoparticle (e.g., EV) surface molecular markers by capturingantibodies labeled with multiple different fluorophores. Such a panel ofantibody capture beads can provide efficient analyses of multiplenanoparticle-associated molecular markers, while serving as spectralreference particles to correct for spectral mixing between thedifferent. The spectral data stream can have the background Rayleigh andRaman scatter spectra subtracted in real time and data can be analyzedin the conventional mode, where signals from various spectral bands areplotted as intensity histograms, or as a hyperspectral data set thatallows spectral approaches that can produce higher resolutionmeasurements.

Example 8 Characterized Liposomes for Detection and/or Sizing ofVesicle-Associated Molecular Markers

Liposomes, i.e., synthetic lipid vesicles, were prepared with definedcompositions and nanoparticle size distributions that can be measured byindependent methods (NTA, RPS, TEM), to serve as reference particles forthe analysis of EVs. The liposomes were found to be stable for months at4° C. FIG. 7 depicts flow cytometry bivariate histograms showingdi-8-ANEPPS vs. Brilliant Violet-conjugated Annexin V fluorescence ofliposomes with or without phosphatidylserine (PS), which binds toannexin V. The liposomes were prepared by extrusion as described inExample 1, resulting in a preparation with a mean diameter of 150 nm, asmeasured by NTA. While both liposome populations (PS− and PS+) showcomparable di-8-ANEPPS fluorescence, the histogram shows enhancedBrilliant Violet-conjugated Annexin V fluorescence in the liposomepopulation containing phosphatidylserine (PS+). Thus, in accordance withthe methods provided herein, fluorescence flow cytometry could be usedto separate distinct populations of nano-liposomes (size in the nmrange), based on the presence or absence of PS as a marker.

Example 9 Supported Bilayer Lipospheres for Standardization of Membrane(Surface Area) Probes

Sub-micron sized silica lipospheres bearing a supported lipid bilayerwere developed for detection by light scatter or for staining by surfacearea probe membrane dyes such as Di-8-ANEPPS and annexin V. 500 nmsilica nanospheres were coated with a lipid bilayer, generating silicalipospheres. The 500 nm silica beads (nanospheres) were obtained fromDuke Scientific (#8050) and sonicated to disperse the beads. 25 μL of astock solution of the beads (3×10¹¹ bead particles/ml) was pipetted into1 mL of 10 mM HEPES buffer pH 7.3, 150 mM NaCl. To the resulting dilutedsolution, 100 μM MLV stock, prepared as described in the section“Preparation of Liposomes” in Example 1 and pre-warmed to 45° C., wasadded. The mixture was vortexed and incubated at 45° C. for 15 minutes,then overnight at ambient temperature on a rotator. The overnightincubation can be performed at any temperature that is above the lipidphase transition temperature—for example, 20-25° C. for some particlelipid compositions, 30-37° C. for other particle lipid compositions.

The next day, the bilayer coated silica beads (nanospheres) were washed3 times in buffered saline, followed by centrifugation. The resultingsilica liposphere preparation was stored at 4° C., at a concentration of1×10⁹ bead particles/ml.

The silica lipospheres can serve as both positive staining controls andspectral compensation reference particles. Presented in FIG. 8 are datafrom lipospheres stained with either Di-8-ANEPPS or DyLight488-annexin Vand analyzed on the custom flow cytometer constructed as described inExample 1, using side scatter as a detection trigger. FIG. 8 shows twodistinct liposphere populations, the population stained with di-8-ANEPPSshowing enhanced di-8-ANEPPS fluorescence and the population stainedwith DyLight488-Annexin V showing enhanced annexin V fluorescence. Theresults demonstrate that both dyes are capable of binding to thelipospheres, thereby validating their use as reference particles.

Example 10 Determining Membrane Dye Saturation using FluorescenceSpectral Shift

Estimates of the concentration and size of vesicles is most accuratewhen the fluorescent probe labeling of the vesicle is close to or atsaturation. We have observed that the fluorescence spectrum of di-8-ANEPPS associated with a vesicle undergoes a spectral shift as itapproaches saturation in the vesicle membrane. Monitoring the spectralshift can be used to determine when probe saturation is reached.

FIGS. 9A and 9B are fluorescence spectra of bulk suspensions of di-8-ANEPPS (500 nM) in buffer alone (HBS; 150 mM NaCl, 10 mM HEPES pH 7.4) orbuffer plus two concentrations of synthetic lipid vesicles (50 uM and 3uM, prepared as in Example 1 above) to produce two different probe tolipid ratios (0.01 and 0.16, respectively). At the lower vesicleconcentration, the probe to lipid ratio is higher and the fluorescenceemission maximum is shifted shifts towards the red. This spectral shiftcan be expressed as the ratio of fluorescence intensity at 690 nm to thefluorescence intensity at 610 nm, and can be used to monitor the probeas it approaches saturation in the membrane. FIG. 9B is a normalizedrepresentation of the measurements depicted in FIG. 9A. Presented inFIG. 9C is the ratio of intensities at 690 to 610 nm measured at severaldifferent probe to lipid ratios. The measurements depicted in FIGS. 9A-Cwere performed in a fluorimeter on bulk solutions in containers, such ascuvettes.

The spectral shift also can be monitored by flow cytometry, using theratio of fluorescence intensity measured through a 690/50 nm band passfilter to the intensity measured through a 610/20 nm bandpass filter.Flow cytometry permits the analysis of individual particles/vesicles.Presented in FIG. 9D are histograms of the population distributions ofthe ratio of intensities of the synthetic vesicles measured through the690/50 nm and 610/20 nm filters (690/610 ratio), for high (0.16) and low(0.01) probe to lipid ratios. The vesicles stained with the higher probeto lipid ratio have a higher 690/610 ratio, which indicates a higherdegree of saturation. FIG. 9E presents the ratio of intensities at 690to 610 nm measured by flow cytometry of synthetic vesicle preparationshaving several different probe to lipid ratios.

The spectral shift also was shown to occur in EVs in plasma. Serialdilutions of platelet-poor plasma (PPP) in buffer were prepared andstained with 500 nM di-8-ANEPPS, to produce samples with different probeto lipid ratios. FIG. 9F presents the median 690/610 ratio at twodilutions of human PPP. The more diluted PPP preparation (1:1600dilution), which has a higher amount of probe relative to the plasmaparticles (higher P/L ratio), showed a spectral shift relative to theless diluted (more concentrated) PPP preparation, as seen by an increasein the 690/610 ratio. Thus, monitoring the 690/610 ratio provides ameans to measure when probe saturation is approached in syntheticvesicle staining as well staining of biological vesicular preparations.

Example 11 Detection of Vesicles and Measurement of their Light Scatter

Flow cytometry of cells generally employs light scatter to triggerdetection, and most examples of vesicle measurements by flow cytometryalso use this approach, which presents a number of difficultiesincluding, but not limited to: 1) the very dim light scatter signalsproduced by vesicle owing to their small size and low refractive index;2) background light scatter from particles in sample, reagents, buffers,and sheath fluids as well as scatter from the flow cell and otheroptical components; 3) differentiation between the dim scatter fromvesicles as discussed in 1) and the various sources of background asdiscussed in 2); and 4) interpreting light scatter intensity given itscomplex dependence on illumination wavelength, particle size (radius),and refractive index. The fluorescence-based detection approach providedherein, which uses a fluorogenic membrane probe, obviates many of theseissues by selectively detecting membrane vesicles with fluorescenceintensity that is proportional to vesicle size (surface area) andrendering light scatter a more useful measurement parameter.

To demonstrate the effectiveness of the fluorescence-based detectionapproach, we analyzed the di-8-ANEPPS fluorescence and violet (405 nm)light scatter in samples of buffer only (HBS; 150 mM NaCl, 10 mM HEPESpH 7.4), buffer plus fluorogenic membrane probe, and buffer plus probeand synthetic vesicles. As presented in FIG. 10A, analysis of bufferalone only produces a small number of background events, a population oflow scattering, low fluorescence events, and a population of higherscatter, higher fluorescence events. Analysis of buffer plus probeproduces a similar number and type of events (FIG. 10B). Analysis ofstained synthetic vesicles (liposomes) produces a distinct population offluorescent events that have light scatter that increases withincreasing fluorescence intensity and estimated diameter (FIG. 10C).When the sample of stained synthetic vesicles is treated with detergent(0.1% Triton X-100; TX1000) to disrupt the membrane vesicles, thedistinct population of fluorescent events is eliminated (FIG. 10D),confirming that this population of events are detected due to thepresence of membrane vesicles. Thus, the particle analysis methodsprovided herein can resolve membrane vesicles.

To further demonstrate the effectiveness of the methods provided herein,stained platelet-free plasma was analyzed, which produced a distinctpopulation of events in which the scatter intensity increased withincreasing fluorescence and estimated diameter (FIG. 10E). The scatterintensity of this population was higher that the light scatter from thestained vesicles/liposomes demonstrated in FIG. 10C, likely owing to thehigher refractive index of cell-derived vesicles in plasma that containproteins, nucleic acids, and other cellular components compared tosynthetic liposomes/vesicles, which are composed only of lipids andbuffer. Addition of detergent eliminates the population of eventsderived from the vesicles in platelet-free plasma (FIG. 10F), confirmingthat it is vesicular in nature. Thus, vesicle flow cytometry usingfluorescence detection addresses many of the difficult issues associatedwith light scatter-based detection, and renders light scattermeasurements more useful for the study of membrane vesicles in complexbiological fluids such as plasma.

Example 12 Multimarker analysis of EVs in Human Plasma

Vesicles in complex biological fluids such as plasma are heterogeneous,originating from different cell types and from different compartmentswithin cells. To demonstrate that the methods provided hereineffectively resolve heterogeneity in these vesicles by measuringmultiple fluorophores simultaneously on individual particles, weprepared human platelet rich plasma (PRP) by centrifuging blood at 50×gfor 15 minutes and collecting the supernatant. The PRP was aged for 24hours, and then centrifuged twice at 2500×g for 15 minutes to preparecell free plasma (PRP supernatant), which was then frozen. Aliquots ofplasma were thawed and stained with di-8-ANEPPS (500 nM) and threedifferent fluorescence-labeled reagents: PECy7-annexin V (a marker ofphosphatidyl serine), PE-anti-CD41 (a marker of CD41 on platelets), andAPC-anti-CD235 (a marker of CD235 on erythrocytes). Annexin V has aspecific binding affinity for phosphatidyl serine (PS), which is asurface molecular marker of many cell-derived EVs, membrane vesicles andliposomes. Synthetic liposomes (prepared as in Example 1 above) bearingphosphatidylserine were also stained using PECy7-annexin V as a positivecontrol and PE-anti-CD41 and APC-anti-CD235 as negative controls, sincethey do not contain the CD41 and CD235 antigens. Intensity calibrationbeads for PE and APC were used to calibrate these signals in units ofmean equivalent soluble fluorochromes (MESF).

Presented in FIG. 11A is the population size distribution of thesynthetic liposomes as estimated from di-8-ANEPPS staining intensity andbivariate histograms of diameter, versus fluorescence intensity of thethree different fluorescence-labeled reagents. The liposomes showedstrong staining for annexin V (PECy7 Intensity) and low levels ofbackground from the PE-anti-CD41 (PE Intensity) and APC-anti-CD235 (APCIntensity), likely resulting from antibody aggregates. Treatment of thesample with the detergent Triton X-100 (0.05%) eliminates the liposomes,while the antibody aggregates and other non-membrane background eventsremain (FIG. 11B). Presented in FIG. 11C is the population sizedistribution of EVs in plasma as estimated from di-8-ANEPPS stainingintensity and bivariate histograms of diameter, versus fluorescenceintensity of the three different fluorescence-labeled reagents. Subsetsof EVs showed staining for CD41 (PE Intensity) and annexin V (PE-Cy7Intensity). When this sample was treated with 0.05% Triton X-100, theEVs were eliminated leaving behind the detergent-insoluble backgroundparticles FIG. 11D). Thus, this method allows the selective detection ofmembrane vesicles, estimation of their size, and measurement of surfacemolecule markers directly in plasma.

Example 13 Examples of Certain Non-Limiting Embodiments

Listed hereafter are non-limiting examples of certain embodiments of thetechnology.

A1. A method of analyzing particles in a sample, the method comprising:

-   -   (a) contacting a sample comprising the particles with one or        more optically detectable labels, thereby forming a staining        solution, wherein:        -   (i) the one or more optically detectable labels comprise a            surface area probe or volume probe, wherein the surface area            probe or volume probe interacts with the particles            stoichiometrically with respect to particle surface area or            volume, respectively, thereby forming particles comprising            particle-associated surface area probe or volume probe,            wherein the optical signal from the particle-associated            surface area probe or volume probe is proportional to the            surface area or volume, respectively, of the particle,            and/or        -   (ii) the one or more optically detectable labels comprise a            molecular marker-specific probe, wherein the molecular            marker-specific probe interacts with a molecular marker of            the particle stoichiometrically with respect to the number            of molecules of the molecular marker that are associated            with the particle, thereby forming particles comprising            particle-associated molecular marker-specific probe, wherein            the optical signal from the particle-associated molecular            marker-specific probe is proportional to the number of            molecules of molecular marker associated with the particle;            and    -   (b) without physical separation or isolation of the particles,        detecting the optical signal of the one or more        particle-associated optically detectable labels generated in (i)        and/or (ii), thereby analyzing the particles in the sample.

A2. The method of embodiment A1, wherein at least one particle in thesample comprises a size of about 500 nm or less in diameter.

A3. The method of embodiment A1 or embodiment A2, wherein at least oneparticle in the sample comprises a size of between about 10 nm to about200 nm in diameter.

A4. The method of any one of embodiments A1 to A3, wherein at least oneparticle in the sample comprises a size of between about 50 nm to about200 nm in diameter.

A5. The method of any one of embodiments A1 to A4, wherein at least oneparticle in the sample comprises a size of between about 50 nm to about150 nm in diameter.

A6. The method of any one of embodiments A1 to A5, wherein the particlesin the sample comprise a size range of between about 10 nm to about 500nm in diameter.

A7. The method of any one of embodiments A1 to A6, wherein the particlesin the sample comprise a size range of between about 50 nm to about 200nm in diameter.

A8. The method of any one of embodiments A1 to A7, wherein the particlesin the sample comprise a size range of between about 50 nm to about 150nm in diameter.

A9. The method of any of embodiments A1 to A8, wherein prior to (a), theconcentration of the particles in the sample is, or is adjusted to,between about 1×10⁶ particles/μL to about 1×10¹² particles/μL.

A10. The method of embodiment A9, wherein the concentration of theparticles in the sample is, or is adjusted to, between about 1×10⁸particles/μL to about 1×10¹⁹ particles/μL

A11. The method of embodiment A10, wherein the concentration of theparticles in the sample is, or is adjusted to, about 1×10⁹ particles/μL.

A12. The method of any of embodiments A1 to A11, wherein the stainingsolution comprises an isotonic buffer.

A13. The method of embodiment A11, wherein the isotonic buffer isphosphate buffered saline (PBS), Hanks balanced salt solution (HBSS) orHEPES buffered saline.

A14. The method of any of embodiments A1 to A13, wherein the stainingsolution comprises a surfactant.

A15. The method of any of embodiments A1 to A14, wherein theconcentration of the surfactant in the staining solution is betweenabout 0.005% to about 0.1%.

A16. The method of any of embodiments A1 to A15, wherein analyzing theparticles in the sample comprises detecting the particles in the sample.

A16.1 The method of any of embodiments A1 to A16, wherein analyzing theparticles in the sample comprises determining the surface area or volumeof the particle based on the detected optical signal of the surface areaprobe or volume probe, respectively.

A16.2 The method of embodiment A16.1, further comprising determining thesize of the particle based on the surface area or volume.

A16.3 The method of embodiment A16.2, wherein determining the size ofthe particle comprises determining the diameter of the particle.

A17. The method of any of embodiments A1 to A16.3, wherein analyzing theparticles in the sample comprises determining the type and/or number ofmolecular markers associated with the particle based on the detectedoptical signal of the molecular marker-associated probe.

A17.1 The method of embodiment A17, further comprising identifyingand/or quantifying the particle based on the type and/or number ofmolecular markers associated with the particle.

A18. The method of any of embodiments A1 to A17, wherein the surfacearea probe or volume probe is a fluorescent label.

A19. The method of any of embodiments A1 to A18, wherein the molecularmarker-specific probe is a fluorescent label.

A20. The method of embodiment A18 or A19, wherein the fluorescent labelis a fluorophore, a tandem conjugate between more than one fluorophore,a fluorescent polymer, a fluorescent protein, or a fluorophoreconjugated to a molecule that interacts with the particle.

A21. The method of any of embodiments A18 to A20, wherein thefluorescent label is conjugated to a molecule that interacts with theparticle.

A22. The method of embodiment A21, wherein the molecule that interactswith the particle is a protein, an antibody, a lectin, a peptide, anucleic acid, a carbohydrate or a glycan.

A23. The method of any of embodiments A1 to A22, wherein at least oneparticle comprises a lipid bilayer.

A24. The method of embodiment A23, wherein the particle comprising alipid bilayer is a liposome or an extracellular vesicle.

A25. The method of any of embodiments A18 to A24, wherein detection ofthe optically detectable label is by fluorescence spectroscopy,fluorescence imaging, or flow cytometry.

A26. The method of embodiment A25, wherein detection of the opticallydetectable label is by flow cytometry.

A27. The method of embodiment A26, wherein the particle is a liposome oran extracellular vesicle.

A28. The method of embodiment A27, wherein the surface area probe isselected from among di-8-ANEPPS, di-4-ANEPPS, a carbocyanine dye or aPKH dye.

A29. The method of embodiment A28, wherein the surface area probe isdi-8-ANEPPS.

A30. The method of any of embodiments A27 to A29, wherein the ratio ofthe amount surface area probe (P) relative to the amount of lipid (L) inthe particle, P/L, is adjusted whereby the surface area probe interactswith the particles stoichiometrically with respect to particle surfacearea or volume.

A31. The method of embodiment A30, wherein the P/L ratio is betweenabout 0.1 to about 0.25.

A32. The method of any of embodiments A27 to A30, wherein the molecularmarker-specific probe is a fluorophore conjugated to a protein

A32.1 The method of embodiment A32, wherein the protein is selected fromamong annexin V, cholera toxin B-subunit, anti-CD61, anti-CD171,anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvIII, anti-EGFR,anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41, anti-CD235,anti-CD54 and anti-CD45.

A33. The method of embodiment A28, A32 or A32.1, wherein the fluorophoreconjugated to the protein conjugates is selected from among Dylight488,a Brilliant Violet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515,PE, rhodamine, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 andAPC-Alexa750.

A34. The method of any of embodiments A1 to A33, wherein physicalseparation or isolation of the particles comprises washing of theparticles.

A35. The method of any of embodiments A1 to A34, wherein physicalseparation or isolation of the particles comprises centrifugation orultracentrifugation of the particles.

A36. The method of any of embodiments A26 to A35, wherein the flowcytometer has a configuration whereby light is collected from both sidesof the flow cell.

A37. The method of any of embodiments A26 to A36, wherein the detectionrange of the flow cytometer is between about 500 fluorescent moleculesper particle to about 5000 fluorescent molecules per particle.

A38. The method of any of embodiments A26 to A37, wherein thefluorescence resolution (R) of the flow cytometer is about or less than200 molecules fluorescein isothiocyanate (FITC).

A39. The method of embodiment A38, wherein R is between about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 moleculesof FITC to about 50, 100 or 150 molecules of FITC.

A40. The method of any of embodiments A1 to A39, wherein:

the particle is an extracellular vesicle; and based on the detectedoptical signal of the molecular marker-specific probe, the type ofmolecular marker associated with the extracellular vesicle isdetermined.

A40.1 The method of embodiment A40, further comprising identifying thecell and/or tissue of origin of the extracellular vesicle based on thetype of molecular marker associated with the extracellular vesicle.

A41. The method of any of embodiments A1 to A40, wherein the samplecomprises a plurality of particles and the method is for determining thesize distribution of the plurality of particles.

A42. The method of embodiment A41, wherein the particles areextracellular vesicles.

A43. The method of any of embodiments A1 to A42, wherein the interactionof the surface area or volume probe and/or the molecular marker-specificprobe with the particle is saturable, whereby the optical signal fromthe surface area probe or volume probe is proportional to the surfacearea or volume, respectively, of the particle and/or the optical signalfrom the molecular marker-specific probe is proportional to the numberof molecules of molecular marker associated with the particle.

B1. A method of detecting, identifying, quantifying and/or determiningthe size of at least a first nanoparticle species in a sample comprisingat two distinct nanoparticle species, the method comprising:

-   -   (a) contacting a sample comprising at least two distinct        nanoparticle species, wherein the distinct nanoparticle species        differ from one another by size and/or by least one molecular        marker associated with each nanoparticle species, with one or        more optically detectable labels comprising a surface area probe        or volume probe, wherein the surface area probe or volume probe        interacts with at least a first nanoparticle species        stoichiometrically with respect to nanoparticle surface area or        volume, respectively, thereby forming particles comprising        particle-associated surface area probe or volume probe, wherein        the optical signal from the particle-associated surface area        probe or volume probe is proportional to the surface area or        volume, respectively, of the first nanoparticle species; and/or    -   (b) contacting the sample with one or more optically detectable        labels comprising a molecular marker-specific probe, wherein the        molecular marker-specific probe interacts with a molecular        marker of at least the first nanoparticle species        stoichiometrically with respect to the number of molecules of        the molecular marker that are associated with the nanoparticle,        thereby forming particles comprising particle-associated        molecular marker-specific probe, wherein the optical signal from        the particle-associated molecular marker-specific probe is        proportional to the number of molecules of the molecular marker        that are associated with the first nanoparticle species;    -   (c) detecting an optical signal from the particle-associated        surface area probe or volume probe and/or detecting an optical        signal from the particle-associated molecular marker-specific        probe, thereby obtaining an optical signal intensity from the        particle-associated surface area or volume probe and/or the        particle-associated molecular marker-specific probe;    -   (d) based on the optical intensity of the particle-associated        surface area probe or volume probe obtained in (c), determining        the surface area or volume, respectively, of at least the first        nanoparticle species, thereby detecting and/or determining the        size of at least the first nanoparticle species in the sample;        and/or    -   (e) based on the optical intensity of the particle-associated        molecular marker-specific probe obtained in (c), determining the        type and/or number of molecular markers associated with at least        the first nanoparticle species, thereby detecting, identifying        and/or quantifying at least the first nanoparticle species in        the sample.

B2. The method of embodiment B1, wherein between at least about 2nanoparticle species to about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 85, 95, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950,975, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000,6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 or more differentnanoparticle species are detected, identified, quantified and/or theirsizes determined.

B3. The method of embodiment B1 or B2, wherein between about 1 to about25 different optically detectable labels are contacted with the samplein (a) and/or between about 1 to about 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 95, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900,925, 950, 975 or 1000 or more different optically detectable labels arecontacted with the sample in (b).

B4. The method of embodiment B3, wherein between about 2 to about 12different optically detectable labels are contacted with the sample in(a) and/or between about 2 to about 12 different optically detectablelabels are contacted with the sample in (b).

B5. The method of any of embodiments B1 to B4, wherein detection of theoptically detectable label is by fluorescence spectroscopy, fluorescenceimaging, or flow cytometry.

B6. The method of embodiment B5, wherein detection of the opticallydetectable label is by flow cytometry.

B7. The method of any of embodiments B1 to B6, wherein the nanoparticleis a liposome or an extracellular vesicle.

B8. The method of embodiment B6 or B7, wherein the surface area probe isselected from among di-8-ANEPPS, di-4-ANEPPS, a carbocyanine dye, a PKHdye or F2N12S.

B9. The method of embodiment B8, wherein the surface area probe isdi-8-ANEPPS.

B10. The method of any of embodiments B7 to B9, wherein the ratio of theamount surface area probe (P) relative to the amount of lipid (L) in theparticle, P/L, is adjusted whereby the surface area probe or volumeprobe interacts with the particles stoichiometrically with respect toparticle surface area or volume, respectively.

B11. The method of embodiment B10, wherein the P/L ratio is betweenabout 0.1 to about 0.25.

B12. The method of any of embodiments B6 to B11, wherein the molecularmarker-specific probe is a fluorophore conjugated to a protein.

B12.1 The method of embodiment B12, wherein the protein is selected fromamong annexin V, cholera toxin B-subunit, anti-CD61, anti-CD171,anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvIII, anti-EGFR,anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41, anti-CD235,anti-CD54 and anti-CD45.

B13. The method of embodiment B8, B12 or B12.1, wherein the fluorophoreconjugated to the protein conjugates is selected from among Dylight488,a Brilliant Violet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515,PE, rhodamine, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 andAPC-Alexa750.

B14. The method of any of embodiments B1 to B13, further comprising,before (c):

-   -   (i) contacting the sample with an optical standard particle        corresponding to at least the first nanoparticle species,        wherein the optical standard particle comprises an optically        detectable label that is the surface area or volume probe that        interacts with the first nanoparticle species in (a) or the        optical standard particle comprises an optically detectable        label that is the molecular marker-specific probe that interacts        with the first nanoparticle species in (b);    -   (ii) detecting an optical signal from the optically detectable        label associated with the optical standard particle in (c),        thereby obtaining an optical signal intensity; and    -   (iii) based on the optical signal intensity obtained in (ii),        adjusting the optical signal intensity of the corresponding        detected optical signal from the particle-associated surface        area or volume probe of the first nanoparticle species and/or        the molecular marker-specific probe of the first nanoparticle        species; and    -   (iv) based on the adjusted optical signal intensity obtained in        (iii), detecting and/or determining the size, type or identity        of the first nanoparticle species in the sample.

B15. The method of any of embodiments B1 to B14, further comprising,before (c):

-   -   (i) contacting the sample with a optical standard particle,        wherein the optical standard particle does not comprise an        optically detectable label;    -   (ii) detecting an optical signal from the optical standard        particle, thereby obtaining an optical signal intensity; and    -   (iii) based on the optical signal intensity obtained in (ii),        adjusting the optical signal intensity of the        particle-associated surface area probe or volume probe of the        first nanoparticle species and/or the molecular marker-specific        probe of the first nanoparticle species; and    -   (iv) based on the adjusted optical signal intensity obtained in        (iii), detecting and/or determining the size, type or identity        of the first nanoparticle species in the sample.

B16. The method of embodiment B14, wherein the optical standard particlecomprising the optically detectable label further comprises an antibody,a liposome or a silica particle.

B17. The methods of any of embodiments B14 to B16, wherein the size ofthe optical standard particle is between about 50 nm to about 500 nm.

B18. The method of embodiment B16 or B17, wherein the optical standardparticle comprising the optically detectable label further comprises anantibody.

B18.1 The method of embodiment B18, wherein the antibody is selectedfrom among anti-CD171, anti-CD325, anti-CD130, anti-GLAST,anti-EGFRvIII, anti-EGFR, anti-CD133, anti-CD15, anti-CD63, anti-CD9,anti-CD41, anti-CD235, anti-CD54, anti-CD45 and anti-IgG.

B19. The method of embodiment B18, wherein the antibody is conjugated toan optically detectable label that is a fluorophore.

B20. The method of embodiment B19, wherein the fluorophore is selectedfrom among DyLight488, a Brilliant Violet dye, Pacific Blue, ChromeOrange, Brilliant Blue 515, PE, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647,APC-Alexa700 and APC-Alexa750.

B21. The method of embodiment B15, wherein the optical standard particlecomprises BSA.

B22. The method of any of embodiments B1 to B21, wherein thenanoparticles comprise nanovesicles.

B23. The method of any of embodiments B1 to B22, wherein thenanovesicles are extracellular vesicles.

B24. The method of embodiment B23, wherein identifying the type oridentity of the first nanoparticle species comprises determining thecellular origin of the extracellular vesicle.

B25. The method of embodiment B24, wherein the cellular origin of theextracellular vesicle is a cancer cell.

B26. The method of embodiment B25, wherein the cancer cell is aglioblastoma cell.

B27. The method of any of embodiments B1 to B26, wherein the size of thenanoparticle species is between about 25 nm to about 900 nm.

B28. The method of embodiment B27, wherein the size of the nanoparticlespecies is between about 100 nm to about 500 nm.

B29. The method of any of embodiments B14 to B17 and B22 to B28, whereinthe optical standard particle comprising the optically detectable labelcomprises a silica particle in association with a lipid bilayer.

B30. The method of any of embodiments B14 to B17 and B22 to B28, whereinthe optical standard particle comprising the optically detectable labelcomprises a liposome.

B31. The method of embodiment B29 or B30, wherein the size of theoptical standard particle is between about 50 nm to about 500 nm.

B32. The method of embodiment B31, wherein the size of the opticalstandard particle is between about 100 nm to about 200 nm.

B33. The method of any of embodiments B29 to B32, wherein the opticalstandard particle comprises an optically detectable label that is asurface area probe.

B34. The method of embodiment B33, wherein the surface area probe is afluorophore selected from among di-8-ANEPPS, di-4-ANEPPS, a carbocyaninedye and a PKH dye.

B35. The method of embodiment B34, wherein the surface area probe isdi-8-ANEPPS.

B36. The method of any of embodiments B6 to B35, wherein the flowcytometer has a configuration whereby light is collected from both sidesof the flow cell.

B37. The method of any of embodiments B6 to B36, wherein the detectionrange of the flow cytometer is between about 500 fluorescent moleculesper particle to about 5000 fluorescent molecules per particle.

B38. The method of any of embodiments B6 to B37, wherein thefluorescence resolution (R) of the flow cytometer is about or less than200 molecules fluorescein isothiocyanate (FITC).

B39. The method of embodiment B38, wherein R is between about 20molecules FITC to about 150 molecules FITC.

B40. The method of any of embodiments B1 to B39, wherein the interactionof the surface area or volume probe and/or the molecular marker-specificprobe with the particle is saturable, whereby the optical signal fromthe surface area probe or volume probe is proportional to the surfacearea or volume, respectively, of the particle and/or the optical signalfrom the molecular marker-specific probe is proportional to the numberof molecules of molecular marker associated with the particle.

C1. A method of determining the size of a nanoparticle of interest in asample using an optically detectable label, the method comprising:

-   -   (a) contacting a nanoparticle of interest with an optically        detectable label comprising a surface area probe or volume        probe, wherein the optically detectable label interacts with the        nanoparticle of interest, whereby a nanoparticle of interest        comprising nanoparticle of interest-associated optically        detectable label is obtained;    -   (b) detecting the nanoparticle of interest-associated optically        detectable label of (a), thereby obtaining an optical signal        intensity;    -   (c) obtaining a predetermined correlation between optical signal        intensity and size of each nanoparticle of a preparation of        nanoparticles comprising a distribution of sizes, wherein:    -   (i) the preparation of nanoparticles is contacted with the        optically detectable label used in (a);    -   (ii) the optically detectable label interacts stoichiometrically        with each of the nanoparticles of the preparation, whereby        nanoparticles comprising nanoparticle-associated optically        detectable label are obtained, wherein the optical signal from        each nanoparticle-associated optically detectable label is        proportional to the surface area or volume of its corresponding        associated nanoparticle;    -   (iii) the optical signals of the nanoparticle-associated        optically detectable labels of (ii) are detected, thereby        obtaining optical signal intensities corresponding to the        nanoparticle-associated optically detectable labels associated        with each nanoparticle of the preparation; and    -   (iv) the optical signal intensity of each        nanoparticle-associated optically detectable label obtained        in (iii) is correlated with the size of its corresponding        associated nanoparticle.; and    -   (d) based on the predetermined correlation obtained according to        (c), and based on the optical signal intensity obtained in (b),        determining the size of the nanoparticle of interest.

C2. The method of embodiment C1, wherein obtaining a correlation in (c)comprises:

-   -   (1) obtaining a preparation of nanoparticles comprising a        distribution of sizes, wherein the preparation does not comprise        the nanoparticle of interest;    -   (2) determining the size distribution of the preparation of        nanoparticles without contacting the preparation with an        optically detectable label;    -   (3) contacting the preparation with an optically detectable        label, wherein the optically detectable label comprises the        surface area probe or volume probe in (a), wherein the surface        area probe or volume probe interacts with the nanoparticles        stoichiometrically with respect to nanoparticle surface area or        volume, respectively, whereby the optical signal from the        optically detectable label is proportional to the surface area        or volume, respectively, of each nanoparticle in the        preparation;    -   (4) detecting the optical signals obtained by (3), thereby        obtaining the optical signal intensities of each nanoparticle in        the preparation; and    -   (5) correlating the optical signal intensities obtained in (4)        with the size distribution determined in (2).

C3. The method of embodiment C1 or C2, wherein the nanoparticle ofinterest is an extracellular vesicle.

C4. The method of any of embodiments C1 to C3, wherein the preparationof nanoparticles comprises liposomes.

C5. The method of any of embodiments C1 to C3, wherein the preparationof nanoparticles comprises silica particles, wherein each silicaparticle comprises a lipid bilayer.

C6. The method of any of embodiments C1 to C5, further comprising addingthe preparation of nanoparticles, whose predetermined correlation hasbeen obtained according to (c), to (a), wherein:

-   -   (1) the optically detectable label interacts with the        nanoparticle of interest and with the preparation of        nanoparticles, whereby a nanoparticle of interest comprising        nanoparticle of interest-associated optically detectable label        and nanoparticles comprising nanoparticle-associated optically        detectable label are obtained;    -   (2) optical signal intensities are obtained from the        nanoparticle of interest-associated optically detectable label        and the nanoparticle-associated optically detectable label in        (b); and    -   (3) the size of the nanoparticle of interest is determined        in (d) based on the predetermined correlation obtained according        to (c) and based on the optical signal intensities obtained from        the nanoparticle of interest-associated optically detectable        label and the nanoparticle-associated optically detectable label        in (b).

C7. The method of any of embodiments C1 to C5, further comprising:

-   -   (1) in the absence of the nanoparticle of interest, contacting        the preparation of nanoparticles, whose predetermined        correlation has been obtained according to (c), with the        optically detectable label used in (a), thereby forming a        mixture comprising the preparation of nanoparticles and the        optically detectable label, wherein the optically detectable        label interacts stoichiometrically with each of the        nanoparticles of the preparation, whereby nanoparticles        comprising nanoparticle-associated optically detectable label        are obtained, wherein the optical signal from each        nanoparticle-associated optically detectable label is        proportional to the surface area or volume of its corresponding        associated nanoparticle;    -   (2) adding the mixture of (1) to (b), thereby detecting the        nanoparticle of interest-associated optically detectable label        and the nanoparticle-associated optically detectable label and        obtaining optical signal intensities of the nanoparticle of        interest-associated optically detectable label and the        nanoparticle-associated optically detectable label; and    -   (3) based on the predetermined correlation obtained according        to (c) and based on the optical signal intensities obtained from        the nanoparticle of interest-associated optically detectable        label and the nanoparticle-associated optically detectable label        in (b), determining the size of the nanoparticle of interest in        (d).

C8. The method of any of embodiments C1 to C7, wherein the interactionof the surface area probe or volume probe with the nanoparticle ofinterest is saturable, whereby the optical signal from the surface areaprobe or volume probe is proportional to the surface area or volume ofthe particle.

D1. A method of identifying and/or quantifying a nanoparticle ofinterest in a sample using an optically detectable label, the methodcomprising:

-   -   (a) contacting a nanoparticle of interest with an optically        detectable label comprising a molecular marker-specific probe,        wherein the optically detectable label interacts with a        molecular marker associated with the nanoparticle of interest,        whereby a nanoparticle of interest comprising nanoparticle of        interest-associated optically detectable label is obtained;    -   (b) detecting the nanoparticle of interest-associated optically        detectable label of (a), thereby obtaining an optical signal        intensity;    -   (c) obtaining a predetermined correlation between optical signal        intensity and the number of molecular markers associated with        each nanoparticle of a preparation of nanoparticles, wherein:    -   (i) the preparation of nanoparticles is contacted with the        optically detectable label used in (a);    -   (ii) the optically detectable label interacts stoichiometrically        with each of the nanoparticles of the preparation, whereby        nanoparticles comprising nanoparticle-associated optically        detectable label are obtained, wherein the optical signal from        each nanoparticle-associated optically detectable label is        proportional to number of molecules of the molecular marker on        the corresponding associated nanoparticle;    -   (iii) the optical signals of the nanoparticle-associated        optically detectable labels of (ii) are detected, thereby        obtaining optical signal intensities corresponding to the        nanoparticle-associated optically detectable labels associated        with each nanoparticle of the preparation; and    -   (iv) the optical signal intensity of each        nanoparticle-associated optically detectable label obtained        in (iii) is correlated with the identity and/or quantity of its        corresponding associated nanoparticle; and    -   (d) based on the predetermined correlation obtained in (c), and        based on the optical signal intensity obtained in (b),        identifying and/or quantifying the nanoparticle of interest.

D2. The method of embodiment C1, wherein obtaining a correlation in (c)comprises:

-   -   (1) obtaining a preparation of nanoparticles comprising a        distribution of different numbers of molecules of a molecular        marker associated with each of the nanoparticles, wherein the        molecular marker is the marker associated with the nanoparticle        of interest in (a) and the preparation does not comprise the        nanoparticle of interest;    -   (2) determining the numbers of the molecular markers in each        nanoparticle of the preparation, without contacting the        preparation with an optically detectable label;    -   (3) contacting the preparation with an optically detectable        label, wherein the optically detectable label comprises the        molecular marker-specific probe in (a), wherein the molecular        marker-specific probe interacts with the nanoparticles        stoichiometrically with respect to the number of molecules of        molecular marker associated with each nanoparticle of them        preparation, whereby the optical signal from the optically        detectable label is proportional to the number of molecules of        molecular marker associated with each nanoparticle of the        preparation;    -   (4) detecting the optical signals obtained by (3), thereby        obtaining the optical signal intensities of each nanoparticle in        the preparation; and    -   (5) correlating the optical signal intensities obtained in (4)        with the numbers of the molecular markers determined in (2).

D3. The method of embodiment D1 or D2, wherein the nanoparticle ofinterest is an extracellular vesicle.

D4. The method of any of embodiments D1 to D3, wherein the preparationof nanoparticles comprises liposomes.

D5. The method of any of embodiments D1 to D3, wherein the preparationof nanoparticles comprises beads comprising at least one antibody,wherein the antibody is conjugated to an optically detectable label.

D6. The method of any of embodiments D1 to D5, further comprising addingthe preparation of nanoparticles, whose predetermined correlation hasbeen obtained according to (c), to (a), wherein:

-   -   (1) the optically detectable label interacts with the        nanoparticle of interest and with the preparation of        nanoparticles, whereby a nanoparticle of interest comprising        nanoparticle of interest-associated optically detectable label        and nanoparticles comprising nanoparticle-associated optically        detectable label are obtained;    -   (2) optical signal intensities are obtained from the        nanoparticle of interest-associated optically detectable label        and the nanoparticle-associated optically detectable label in        (b); and    -   (3) the nanoparticle of interest is identified and/or quantified        in (d) based on the predetermined correlation obtained according        to (c) and based on the optical signal intensities obtained from        the nanoparticle of interest-associated optically detectable        label and the nanoparticle-associated optically detectable label        in (b).

D7. The method of any of embodiments D1 to d5, further comprising:

-   -   (1) in the absence of the nanoparticle of interest, contacting        the preparation of nanoparticles, whose predetermined        correlation has been obtained according to (c), with the        optically detectable label used in (a), thereby forming a        mixture comprising the preparation of nanoparticles and the        optically detectable label, wherein the optically detectable        label interacts stoichiometrically with each of the        nanoparticles of the preparation, whereby nanoparticles        comprising nanoparticle-associated optically detectable label        are obtained, wherein the optical signal from each        nanoparticle-associated optically detectable label is        proportional to the surface area or volume of its corresponding        associated nanoparticle;    -   (2) adding the mixture of (1) to (b), thereby detecting the        nanoparticle of interest-associated optically detectable label        and the nanoparticle-associated optically detectable label and        obtaining optical signal intensities of the nanoparticle of        interest-associated optically detectable label and the        nanoparticle-associated optically detectable label; and    -   (3) based on the predetermined correlation obtained according        to (c) and based on the optical signal intensities obtained from        the nanoparticle of interest-associated optically detectable        label and the nanoparticle-associated optically detectable label        in (b), identifying and/or quantifying the nanoparticle of        interest in (d).

D8. The method of any of embodiments D1 to D7, wherein the interactionof the surface area or volume probe and/or the molecular marker-specificprobe with the particle is saturable, whereby the optical signal fromthe surface area probe or volume probe is proportional to the surfacearea or volume, respectively, of the particle and/or the optical signalfrom the molecular marker-specific probe is proportional to the numberof molecules of molecular marker associated with the particle.

E1. A preparation of optical standard particles, comprising a silicaparticle and a lipid bilayer in association with the silica particle,wherein the preparation has a distribution of optical standard particlesizes between about 10 nm to about 900 nm.

E2. The preparation of embodiment E1, wherein the lipid bilayercomprises an outer coating on the silica particle.

E3. The preparation of embodiment E1 or E2, wherein the distribution ofoptical standard particle sizes is between about 50 nm to about 500 nm.

E4. The preparation of embodiment E3, wherein the distribution ofoptical standard particle sizes is between about 20 nm to about 200 nm.

E5. A optical standard particle, comprising a silica particle and alipid bilayer in association with the silica particle.

E6. The optical standard particle of embodiment E5, wherein the lipidbilayer comprises an outer coating on the silica particle.

E7. The optical standard particle of embodiment E5 or E6, wherein thesize of the optical standard particle is about 50 nm to about 200 nm.

E8. The optical standard particle of embodiment E7, wherein the size ofthe optical standard particle is about 100 nm to about 150 nm.

F1. The method of any of embodiments A1 to A43, B1 to B40, C1 to C8 orD1 to D8, wherein the optically detectable label associated with theparticle is detected in the presence of free label.

F2. The method of any of embodiments A1 to A43, B1 to B40, C1 to C8, D1to D8 or F1, wherein the sample is a biological sample.

F3. The method of embodiment F2, wherein the biological sample comprisesparticles and a biological fluid.

F4. The method of embodiment F3, wherein the biological fluid comprisesblood, plasma, serum, urine, saliva, seminal fluid, lavages, cervicalfluid, cervicovaginal fluid, cerebrospinal fluid, vaginal fluid, breastfluid, breast milk, synovial fluid, semen, seminal fluid, sputum,cerebral spinal fluid, tears, mucus, interstitial fluid, follicularfluid, amniotic fluid, aqueous humor, vitreous humor, peritoneal fluid,ascites, sweat, lymphatic fluid, lung sputum or combinations, fractionsor components thereof.

F5. The method of embodiment F4, wherein the biological fluid comprisescerebrospinal fluid.

F6. The method of any of embodiments F2 to F5, wherein the biologicalsample comprises particles derived from a cell or tissue.

F7. The method of embodiment F6, wherein the cell or tissue is a cancercell or tissue.

F8. The method of embodiment F6 or F7, wherein the cell or tissue isselected from among liver, lung, spleen, pancreas, colon, skin, bladder,eye, brain, esophagus, head, neck, ovary, testes, prostate, placenta,epithelium, endothelium, adipocyte, kidney, heart, muscle, blood andcombinations thereof.

F9. The method of embodiment F7 or F8, wherein the sample comprises abiological fluid.

F10. The method of embodiment F9, wherein the biological fluid is blood,plasma, serum, saliva, urine or cerebrospinal fluid.

F11. The method of embodiment F10, wherein the biological fluid issaliva, urine or serum.

F12. The method of embodiment F11, wherein the biological samplecomprises particles derived from a cancer cell or tissue and the canceris ovarian, lung, bladder or prostate cancer.

F13. The method of embodiment F10, wherein the biological fluid iscerebrospinal fluid.

F14. The method of embodiment F13, wherein the biological samplecomprises particles derived from a cancer cell or tissue and the canceris brain cancer.

F15. The method of embodiment F14, wherein the brain cancer isglioblastoma.

F16. the method of any of embodiments F2 to F15, wherein the particle isselected from among membrane vesicles, liposomes, lipoproteins,apoptotic bodies, viruses, viral particles, virus-like particles,extracellular vesicles or combinations thereof.

F17. The method of embodiment F16, wherein the particle is anextracellular vesicle.

F18. The method of embodiment F17, wherein the sample comprises morethan one type of extracellular vesicle, wherein each vesicle originatesfrom a cell or tissue that is different from the cell or tissue oforigin of at least one other type of extracellular vesicle in thesample.

G1. The method of any of embodiments A1 to A43, B1 to B40, C1 to C8, D1to D8 or F1 to F18, wherein large particulates other than the particles,cells, cellular debris or a combination thereof are removed from thesample.

G2. The method of embodiment G1, wherein the large particulates otherthan the particles, cells, cellular debris or a combination thereof areremoved by centrifugation. X1. The method of any of embodiments A1 toA43, B1 to B40, C1 to C8, D1 to D8, F1 to F18, G1 or G2, wherein theamount or concentration of the optically detectable label approaches oris at saturation.

X2. The method of embodiment X1, wherein approaching saturation or beingat saturation is determined by the method of any of embodiments H0 toH54.

H0. A method of determining whether staining of a lipid-containingparticle with an optically detectable label selected from among asurface area probe, a volume probe or a molecular marker-specific probeof a particle is approaching saturation or is saturated, the methodcomprising:

-   -   (a) contacting a first amount of a sample comprising one or more        of the lipid-containing particles with a first concentration of        the probe, thereby forming a first staining solution comprising        the probe associated with the particles;    -   (b) determining the wavelength at which the maximum optical        signal intensity of the particle-associated probe is detected,        thereby obtaining a first maximum optical signal wavelength;    -   (c) obtaining a predetermined second maximum optical wavelength        corresponding to a second staining solution comprising a second        amount of the sample and/or a second concentration of the probe,        or performing (a) and (b) using a second amount of the sample        and/or a second concentration of the probe, wherein the ratio of        the second concentration of the probe relative to the second        amount of the sample is less than the ratio of the first        concentration of the probe relative to the first amount of the        sample; and    -   (d) analyzing the first maximum optical signal wavelength of (b)        and the second maximum optical wavelength of (c), whereby    -   if the first maximum optical signal wavelength is different from        the second maximum optical signal wavelength, then the first        staining solution comprises one or more lipid-containing        particles comprising a ratio of probe to lipid that is        approaching saturation or has saturated.

H1. The method of embodiment HO, further comprising:

-   -   (i) before (a), contacting the sample with a first amount of an        optical standard particle, wherein the optical standard particle        comprises a known amount of lipid;    -   (ii) in (a), contacting the sample comprising the optical        standard particle with the first concentration of probe and        determining the amount of probe relative to the amount of lipid        in the optical standard particle of the first staining solution        formed in (a), thereby obtaining a first probe to lipid ratio,        P/L, of the optical standard particle in the first staining        solution;    -   (iii) in (c), obtaining a predetermined second maximum optical        wavelength corresponding to a second staining solution        comprising a second amount of the optical standard particle        and/or a second concentration of the probe corresponding to a        second probe to lipid ratio, P′/L′, of the optical standard        particle in the second staining solution, or performing (a)        and (b) using a second amount of the optical standard particle        and/or a second concentration of the probe, thereby obtaining a        second probe to lipid ratio, P′/L′, wherein P′/L′ is less than        P/L;    -   (iv) in (d), obtaining a correlation between the probe to lipid        ratios of the optical standard particle in the first and second        staining solutions and the first and second maximum optical        signal wavelengths wherein the first maximum optical signal        wavelength corresponds to the probe to lipid ratio P/L and the        second maximum optical signal wavelength corresponds to the        probe to lipid ratio P′/L′ whereby, if the first maximum optical        signal wavelength is different from the second maximum optical        signal wavelength, the probe to lipid ratio P/L is identified as        a ratio that approaches saturation or is at saturation; and    -   (v) if the probe to lipid ratio P/L is identified as a ratio        that approaches saturation or is at saturation in (iv),        determining that the amount of probe added to the first staining        solution approaches saturation or saturates in the        lipid-containing particle in the first staining solution.

H2. The method of embodiment H0 or H1 that is performed for a pluralityof amounts of sample and/or concentrations of probe, thereby obtaining aplurality of staining solutions each corresponding to a unique ratio ofprobe concentration to sample amount ranging from a lowest ratio to ahighest ratio, or each corresponding to a unique probe to lipid ratioranging from a lowest ratio to a highest ratio, wherein a stainingsolution is identified as comprising a concentration of probe thatapproaches or is at saturation if:

-   -   the maximum optical signal wavelength of the staining solution        is different from the maximum optical signal wavelength of a        second staining solution comprising a lower ratio of probe        concentration to sample amount and/or a lower ratio of probe to        lipid; and/or    -   the maximum optical signal wavelength of the staining solution        is about the same as the maximum optical signal wavelength of a        third staining solution comprising a higher ratio of probe        concentration to sample amount and/or a higher ratio of probe to        lipid.

H3. A method of determining whether staining of a lipid-containingparticle with an optically detectable label selected from among asurface area probe, a volume probe or a molecular marker-specific probeof a particle is approaching saturation or is saturated, the methodcomprising:

-   -   (a) contacting a first amount of a sample comprising one or more        of the lipid-containing particles with a first concentration of        the probe, thereby forming a first staining solution comprising        the probe associated with the particles, wherein the particles        comprise a known amount of lipid;    -   (b) determining the amount of probe relative to the amount of        lipid in the particles of the solution formed in (a), thereby        obtaining a first probe to lipid ratio, P/L;    -   (c) determining the wavelength at which the maximum optical        signal intensity of the probe associated with the particles        in (b) is detected, thereby obtaining a first maximum optical        signal wavelength corresponding to the probe to lipid ratio,        P/L;    -   (d) obtaining a predetermined second maximum optical wavelength        corresponding to a second staining solution comprising particles        that comprise a second probe to lipid ratio, P′/L′, or        performing (a)-(c) using a second amount of the sample and/or a        second concentration of the probe whereby particles comprising a        second probe to lipid ratio, P′/L′, is obtained, wherein P′/L′        is less than P/L; and    -   (e) analyzing the first maximum optical signal wavelength of (c)        and the second maximum optical wavelength of (d), whereby, if        the maximum optical signal wavelength of (c) is different from        the second maximum optical signal wavelength of (d), staining of        the particle with the probe at the ratio P/L is approaching        saturation or has saturated.

H4. The method of any of embodiments H0 to H3, wherein the first maximumoptical signal wavelength is higher than the second maximum opticalsignal wavelength.

H5. The method of any of embodiments H0 to H4, wherein the differencebetween the first maximum optical wavelength and the second maximumoptical wavelength is between about 0.5 nm to about 30, 40, 45, 50, 55,60, 65, 70, 75, 80, 90 or 100 nm.

H6. The method of any of embodiments H0 to H4, wherein the differencebetween the first maximum optical wavelength and the second maximumoptical wavelength is about 0.5 nm, 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 4nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 14 nm, 15 nm, 16 nm, 17nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27nm, 28 nm, 29 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm or 100 nm.

H7. A method of determining whether staining of a lipid-containingparticle with an optically detectable label selected from among asurface area probe, a volume probe or a molecular marker-specific probeof a particle is approaching saturation or is saturated, the methodcomprising:

-   -   (a) contacting a first amount of a sample comprising one or more        of the lipid-containing particles with a first concentration of        the probe, thereby forming a staining solution comprising the        probe associated with the particles;    -   (b) contacting a second amount of the sample comprising one or        more of the lipid-containing particles with a second        concentration of the probe, thereby forming a second staining        solution comprising the probe associated with the particles,        wherein the ratio of the second concentration of the probe        relative to the second amount of the sample is less than the        ratio of the first concentration of the probe relative to the        first amount of the sample;    -   (c) determining the optical signal intensity of the first        staining solution at a first optical wavelength (A1) and at a        second optical wavelength (A2), thereby obtaining a ratio        (C1/C2) of the optical signal intensity at the first optical        wavelength relative to the optical signal intensity at the        second optical wavelength;    -   (d) determining the optical signal intensity of the second        staining solution at the first optical wavelength (A1) and at        the second optical wavelength (A2), thereby obtaining a ratio        (C1′/C2′) of the optical signal intensity at the first optical        wavelength relative to the optical signal intensity at the        second optical wavelength; and    -   (e) analyzing the ratios obtained in (c) and (d), whereby:    -   if C1/C2 is greater than C1′/C2′, then the first staining        solution comprises one or more lipid-containing particles        comprising a ratio of probe to lipid that is approaching        saturation or has saturated.

H8. The method of embodiment H7, further comprising:

-   -   (i) before (a), contacting the sample with a first amount of an        optical standard particle, wherein the optical standard particle        comprises a known amount of lipid;    -   (ii) before (b), contacting the sample with a second amount of        the optical standard particle;    -   (iii) in (a), contacting the sample comprising the optical        standard particle with the first concentration of probe and        determining the amount of probe relative to the amount of lipid        in the optical standard particle of the first staining solution        formed in (a), thereby obtaining a first probe to lipid ratio,        P/L, of the optical standard particle in the first staining        solution;    -   (iv) in (b), contacting the sample comprising the optical        standard particle with the second concentration of probe and        determining the amount of probe relative to the amount of lipid        in the optical standard particle of the second staining solution        formed in (b), thereby obtaining a second probe to lipid ratio,        P′/L′, of the optical standard particle in the second staining        solution, wherein P′/L′ is less than P/L;    -   (v) in (e), obtaining a correlation between the probe to lipid        ratios of the optical standard particle in the first and second        staining solutions and the ratios of the optical signal        intensities at the first optical wavelength relative to the        second optical wavelength whereby, if the ratio of the optical        signal intensity at the first optical wavelength relative to the        optical signal intensity at the second optical wavelength for        the first staining solution is greater than the ratio of the        optical signal intensity at the first optical wavelength        relative to the optical signal intensity at the second optical        wavelength for the second staining solution, then the probe to        lipid ratio P/L is identified as a ratio that approaches        saturation or is at saturation; and    -   (vi) if the probe to lipid ratio P/L is identified as a ratio        that approaches saturation or is at saturation in (v),        determining that the amount of probe added to the first staining        solution approaches saturation or saturates in the        lipid-containing particle in the first staining solution.

H8.1. A method of determining whether staining of a lipid-containingparticle with an optically detectable label selected from among asurface area probe, a volume probe or a molecular marker-specific probeof a particle is approaching saturation or is saturated, the methodcomprising:

-   -   (a) contacting a sample comprising one or more of the        lipid-containing particles with an optical standard particle,        wherein staining of the optical standard particle is        predetermined to approach saturation or saturate at a known        ratio, C1/C2, of optical signal intensity at a first optical        wavelength (A1) relative to the optical signal intensity at a        second optical wavelength (A2);    -   (b) contacting the sample comprising the one or lipid-containing        particles and the optical standard particle with the probe,        thereby forming a staining solution comprising the probe        associated with the lipid-containing particles and the optical        standard particle;    -   (c) determining the optical signal intensity of the staining        solution at a first optical wavelength (A1) and at a second        optical wavelength (A2), thereby obtaining a ratio (C1′/C2′) of        the optical signal intensity at the first optical wavelength        relative to the optical signal intensity at the second optical        wavelength; and    -   (d) analyzing the ratio obtained in (c), whereby:    -   if C1′/C2′ is equal to or equal to about C1/C2, then the        staining solution comprises one or more lipid-containing        particles comprising a ratio of probe to lipid that is        approaching saturation or has saturated.

H9. The method of embodiment H7 or H8 that is performed for a pluralityof amounts of sample and/or concentrations of probe, thereby obtaining aplurality of staining solutions each corresponding to a unique ratio ofprobe concentration to sample amount ranging from a lowest ration to ahighest ratio, or each corresponding to a unique probe to lipid ratioranging from a lowest ratio to a highest ratio, wherein a stainingsolution is identified as comprising a concentration of probe thatapproaches or is at saturation if:

-   -   the ratio of optical signal intensity at the first optical        wavelength relative to the optical signal intensity at the        second optical wavelength of the staining solution is different        from the ratio of optical signal intensity at the first optical        wavelength relative to the optical signal intensity at the        second optical wavelength of a second staining solution        comprising a lower ratio of probe concentration to sample amount        and/or a lower ratio of probe to lipid; and/or    -   the ratio of optical signal intensity at the first optical        wavelength relative to the optical signal intensity at the        second optical wavelength of the staining solution is about the        same as or greater than the ratio of optical signal intensity at        the first optical wavelength relative to the optical signal        intensity at the second optical wavelength of a second staining        solution comprising a higher ratio of probe concentration to        sample amount and/or a higher ratio of probe to lipid.

H10. The method of embodiment H9, wherein the ratio of optical signalintensity at the first optical wavelength relative to the optical signalintensity at the second optical wavelength of the staining solution isgreater than the ratio of optical signal intensity at the first opticalwavelength relative to the optical signal intensity at the secondoptical wavelength of a second staining solution comprising a lowerratio of probe concentration to sample amount and/or a lower ratio ofprobe to lipid.

H11. The method of embodiment H9 or H10, wherein the ratio of opticalsignal intensity at the first optical wavelength relative to the opticalsignal intensity at the second optical wavelength of the stainingsolution is about the same as or greater than the ratio of opticalsignal intensity at the first optical wavelength relative to the opticalsignal intensity at the second optical wavelength of a second stainingsolution comprising a higher ratio of probe concentration to sampleamount and/or a higher ratio of probe to lipid.

H12. A method of determining whether staining of a lipid-containingparticle with an optically detectable label selected from among one ormore of surface area probe, a volume probe or a molecularmarker-specific probe of a particle is approaching saturation or issaturated, wherein the amount of lipid associated with thelipid-containing particle is known, the method comprising:

-   -   (a) contacting a sample comprising one or more of the        lipid-containing particles with a first amount of the sample        comprising one or more of the lipid-containing particles and a        first concentration of the probe, thereby forming a first        staining solution comprising the probe associated with the        particles;    -   (b) contacting the sample comprising one or more of the        lipid-containing particles with a second amount of the sample        comprising one or more of the lipid-containing particles and/or        a second concentration of the probe, thereby forming a second        staining solution comprising the probe associated with the        particles;    -   (c) determining the amount of probe relative to the amount of        lipid in the particles in the first staining solution of (a) and        in the particles in the second staining solution of (b), thereby        obtaining a first probe to lipid ratio, P/L, corresponding to        the first staining solution and a second probe to lipid ratio,        P′/L′, corresponding to the second staining solution;    -   (d) determining the optical signal intensity of the first        staining solution at a first optical wavelength (A1) and at a        second optical wavelength (A2), thereby obtaining a ratio        (C1/C2) of the optical signal intensity at the first optical        wavelength relative to the optical signal intensity at the        second optical wavelength;    -   (e) determining the optical signal intensity of the second        staining solution at the first optical wavelength (A1) and at        the second optical wavelength (A2), thereby obtaining a ratio        (C1′/C2′) of the optical signal intensity at the first optical        wavelength relative to the optical signal intensity at the        second optical wavelength; and    -   (f) analyzing the ratios obtained in (c)-(e), whereby:    -   if P/L is greater than P′/L′ and C1/C2 is greater than C1′/C2′,        staining of the particle with the probe at the ratio P/L is        approaching saturation or has saturated, and    -   if P′/L′ is greater than P/L and C1′/C2′ is greater than C1/C2,        staining of the particle with the probe at the ratio P′/L′ is        approaching saturation or has saturated.

H13. The method of embodiment H12, wherein a plurality of differentamounts of probe is contacted with the sample in (a), thereby obtaininga plurality of solutions each comprising a different probe to lipidratio, and analyzing the ratios in (f) comprises:

-   -   analyzing, for each of the plurality of solutions comprising a        different probe to lipid ratio, a corresponding ratio of optical        signal intensity at the first optical wavelength relative to the        optical signal intensity at the second optical wavelength,        thereby obtaining a plurality of ratios of optical signal        intensities at the first optical wavelength relative to the        optical signal intensity at the second optical wavelength; and    -   from the plurality of ratios, determining the maximum ratio of        optical signal intensity at the first optical wavelength        relative to the optical signal intensity at the second optical        wavelength, whereby the probe to lipid ratio corresponding to        the maximum ratio of optical signal intensity at the first        optical wavelength relative to the optical signal intensity at        the second optical wavelength is the probe to lipid ratio at        which staining of the particle is approaching saturation or has        saturated.

H14. The method of any of embodiments H7 to H13, wherein the firstoptical wavelength is greater than the second optical wavelength.

H15. The method of any of embodiments H0 to H6, wherein the firstmaximum optical wavelength and/or the second maximum optical wavelengthare in the range of between 350 to 950 nm, between 400-900 nm, orbetween 600 to 800 nm.

H16. The method of any of embodiments H7 to H14, wherein the firstoptical wavelength and/or the second optical wavelength are in the rangeof between 350 to 950 nm, between 400 to 900 nm, or between 600 to 800nm.

H17. The method of embodiment H15, wherein the first maximum opticalwavelength and/or the second maximum optical wavelength are in the rangeof between 600 to 750 nm.

H18. The method of any of embodiments H7 to H14, wherein the firstoptical wavelength is in the range of between 680-700 nm and the secondoptical wavelength is in the range of between 600 to 720 nm.

H19. The method of embodiment H18, wherein the first optical wavelengthis 690 nm and the second optical wavelength is 610 nm.

H20. The method of any of embodiments H0 to H19, wherein at least onelipid-containing particle in the sample comprises a size of about 1000nm, 950 nm, 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550nm or 500 nm or less in diameter.

H21. The method of embodiment H20, wherein at least one particle in thesample comprises a size of between about 10 nm to about 200 nm indiameter.

H22. The method of embodiment H21, wherein at least one particle in thesample comprises a size of between about 50 nm to about 200 nm indiameter.

H23. The method of embodiment H22, wherein at least one particle in thesample comprises a size of between about 50 nm to about 150 nm indiameter.

H24. The method of any one of embodiments H0 to H19, wherein thelipid-containing particles in the sample comprise a size range ofbetween about 10 nm to about 500 nm in diameter.

H25. The method of embodiment H24, wherein the particles in the samplecomprise a size range of between about 50 nm to about 200 nm indiameter.

H26. The method of embodiment H25, wherein the particles in the samplecomprise a size range of between about 50 nm to about 150 nm indiameter.

H27. The method of any of embodiments H1, H2, H4-H6, H8-H11 or H14-H26,wherein at least one optical standard particle in the sample comprises asize of about 1000 nm, 950 nm, 900 nm, 850 nm, 800 nm, 750 nm, 700 nm,650 nm, 600 nm, 550 nm or 500 nm or less in diameter.

H28. The method of embodiment H27, wherein at least one particle in thesample comprises a size of between about 10 nm to about 200 nm indiameter.

H29. The method of embodiment H28, wherein at least one particle in thesample comprises a size of between about 50 nm to about 200 nm indiameter.

H30. The method of embodiment H29, wherein at least one particle in thesample comprises a size of between about 50 nm to about 150 nm indiameter.

H31. The method of any one of embodiments H1, H2, H4-H6, H8-H11 orH14-H26, wherein the lipid-containing particles in the sample comprise asize range of between about 10 nm to about 500 nm in diameter.

H32. The method of embodiment H31, wherein the particles in the samplecomprise a size range of between about 50 nm to about 200 nm indiameter.

H33. The method of embodiment H32, wherein the particles in the samplecomprise a size range of between about 50 nm to about 150 nm indiameter.

H34. The method of any of embodiments H0 to H33, wherein prior to (a),the concentration of the lipid-containing particles in the sample is, oris adjusted to, between about 1×10⁶ particles/μL to about 1×10¹²particles/μL.

H35. The method of embodiment H34, wherein the concentration of theparticles in the sample is, or is adjusted to, between about 1×10⁸particles/μL to about 1×10¹⁰ particles/μL

H36. The method of embodiment H35, wherein the concentration of theparticles in the sample is, or is adjusted to, about 1×10⁹ particles/μL.

H37. The method of any of embodiments H0 to H36, wherein the surfacearea probe or volume probe is a fluorescent label.

H38. The method of any of embodiments H0 to H37, wherein the molecularmarker-specific probe is a fluorescent label.

H39. The method of embodiment H37 or embodiment H38, wherein thefluorescent label is a fluorophore, a tandem conjugate between more thanone fluorophore, a fluorescent polymer, a fluorescent protein, or afluorophore conjugated to a molecule that interacts with the particle.

H40. The method of any of embodiments H37 to H39, wherein thefluorescent label is conjugated to a molecule that interacts with theparticle.

H41. The method of embodiment H40, wherein the molecule that interactswith the particle is a protein, an antibody, a lectin, a peptide, anucleic acid, a carbohydrate or a glycan.

H42. The method of any of embodiments H0 to H41, wherein at least onelipid-containing particle comprises a lipid bilayer.

H43. The method of any of embodiments H1, H2, H4-H6, H8-H11 or H14-H42,wherein at least one optical standard particle comprises a lipidbilayer.

H44. The method of embodiment H42 or H43, wherein the particle is aliposome or an extracellular vesicle.

H44.1 The method of any of embodiments H0 to H44, wherein the opticalwavelength and/or intensity is obtained by analyzing the sample in bulk.

H44.2 The method of any of embodiments H0 to H44, wherein the opticalwavelength and/or intensity is obtained by analyzing individualparticles of the sample.

H45. The method of any of embodiments H0 to H44.2, wherein the opticalwavelength and/or intensity is determined by fluorescence spectroscopy,fluorescence imaging, or flow cytometry.

H46. The method of embodiment H45, wherein the optical wavelength and/orintensity is determined by flow cytometry.

H47. The method of embodiment H46, wherein individual particles of thesample are analyzed and the lipid-containing particle is a membranevesicle, liposome, lipoprotein, apoptotic body, virus, viral particle,virus-like particle, extracellular vesicle or a combination thereof.

H48. The method of embodiment H46 or H47, wherein individual particlesof the sample are analyzed and the optical standard particle is amembrane vesicle, liposome, lipoprotein, apoptotic body, virus, viralparticle, virus-like particle, extracellular vesicle or a combinationthereof.

H49. The method of embodiment H48, wherein the probe is a surface areaprobe selected from among di-8-ANEPPS, di-4-ANEPPS, a carbocyanine dyeor a PKH dye.

H50. The method of embodiment H49, wherein the surface area probe isdi-8-ANEPPS.

H51. The method of any of embodiments H38 to H48, wherein the probe is amolecular marker-specific probe comprising a fluorophore conjugated to aprotein

H52. The method of embodiment H51, wherein the protein is selected fromamong annexin V, cholera toxin B-subunit, anti-CD61, anti-CD171,anti-CD325, anti-CD130, anti-GLAST, anti-EGFRvIII, anti-EGFR,anti-CD133, anti-CD15, anti-CD63, anti-CD9, anti-CD41, anti-CD235,anti-CD54 and anti-CD45.

H53. The method of embodiment H51 or H52, wherein the fluorophoreconjugated to the protein is selected from among Dylight488, a BrilliantViolet dye, Pacific Blue, Chrome Orange, Brilliant Blue 515, PE,rhodamine, FITC, PE-Cy5.5, PE-Cy7, APC, Alexa647, APC-Alexa700 andAPC-Alexa750.

H54. The method of any of embodiments H0 to H53, wherein the opticalwavelength and/or intensity is from fluorescence emission, fluorescenceexcitation, fluorescence absorbance, fluorescence anisotropy,fluorescence polarization, fluorescence lifetime, or a combinationthereof.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments disclosed in this application,yet modifications and improvements are within the scope and spirit ofthe technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claims thatfollow:

What is claimed is:
 1. A method of analyzing particles in a sample, themethod comprising: (a) contacting a sample comprising the particles withone or more optically detectable labels, thereby forming a stainingsolution, wherein: (i) the one or more optically detectable labelscomprise a surface area probe or volume probe, wherein the surface areaprobe or volume probe interacts with the particles stoichiometricallywith respect to particle surface area or volume, respectively, therebyforming particles comprising particle-associated surface area probe orparticle-associated volume probe, wherein the optical signal from theparticle-associated surface area or volume probe is proportional to thesurface area or volume, respectively, of the particle, and/or (ii) theone or more optically detectable labels comprise a molecularmarker-specific probe, wherein the molecular marker-specific probeinteracts with a molecular marker of the particle stoichiometricallywith respect to the number of molecules of the molecular marker that areassociated with the particle, thereby forming particles comprisingparticle-associated molecular marker-specific probe, wherein the opticalsignal from the particle-associated molecular marker-specific probe isproportional to the number of molecules of molecular marker associatedwith the particle; and (b) without physical separation or isolation ofthe particles, detecting the optical signal of the one or moreparticle-associated optically detectable labels generated in (i) and/or(ii), thereby analyzing the particles in the sample.
 2. The method ofclaim 1, wherein analyzing the particles in the sample comprisesdetecting the particles in the sample.
 3. The method of claim 1, whereinanalyzing the particles in the sample comprises determining the surfacearea or volume of the particle based on the detected optical signal ofthe surface area probe or volume probe, respectively.
 4. The method ofclaim 3, further comprising determining the size of the particle basedon the surface area or volume.
 5. The method of claim 1, whereinanalyzing the particles in the sample comprises determining the typeand/or number of molecular markers associated with the particle based onthe detected optical signal of the molecular marker-associated probe. 6.The method of claim 5, further comprising identifying and/or quantifyingthe particle based on the type and/or number of molecular markersassociated with the particle.
 7. The method of claim 1, wherein thesurface area probe, volume probe and/or molecular marker-specific probeis a fluorescent label.
 8. The method of claim 7, wherein thefluorescent label is a fluorophore, a tandem conjugate between more thanone fluorophore, a fluorescent polymer, a fluorescent protein, or afluorophore conjugated to a molecule that interacts with the particle.9. The method of claim 1, wherein at least one particle comprises alipid bilayer.
 10. The method of claim 9, wherein the particlecomprising a lipid bilayer is a membrane vesicle, a liposome or anextracellular vesicle.
 11. The method of claim 1, wherein detection ofthe optically detectable label is by fluorescence spectroscopy,fluorescence imaging, or flow cytometry.
 12. The method of claim 1,wherein physical separation or isolation of the particles compriseswashing of the particles.
 13. The method of claim 1, wherein physicalseparation or isolation of the particles comprises centrifugation orultracentrifugation of the particles.
 14. A method of determining thesize of a nanoparticle of interest in a sample using an opticallydetectable label, the method comprising: (a) contacting a nanoparticleof interest with an optically detectable label comprising a surface areaprobe or volume probe, wherein the optically detectable label interactswith the nanoparticle of interest, whereby a nanoparticle of interestcomprising nanoparticle of interest-associated optically detectablelabel is obtained; (b) detecting the nanoparticle of interest-associatedoptically detectable label of (a), thereby obtaining an optical signalintensity; (c) obtaining a predetermined correlation between opticalsignal intensity and size of each nanoparticle of a preparation ofnanoparticles comprising a distribution of sizes, wherein: (i) thepreparation of nanoparticles is contacted with the optically detectablelabel used in (a); (ii) the optically detectable label interactsstoichiometrically with each of the nanoparticles of the preparation,whereby nanoparticles comprising nanoparticle-associated opticallydetectable label are obtained, wherein the optical signal from eachnanoparticle-associated optically detectable label is proportional tothe surface area or volume of its corresponding associated nanoparticle;(iii) the optical signals of the nanoparticle-associated opticallydetectable labels of (ii) are detected, thereby obtaining optical signalintensities corresponding to the nanoparticle-associated opticallydetectable labels associated with each nanoparticle of the preparation;and (iv) the optical signal intensity of each nanoparticle-associatedoptically detectable label obtained in (iii) is correlated with the sizeof its corresponding associated nanoparticle.; and (d) based on thepredetermined correlation obtained according to (c), and based on theoptical signal intensity obtained in (b), determining the size of thenanoparticle of interest.
 15. The method of claim 14, wherein obtaininga correlation in (c) comprises: (1) obtaining a preparation ofnanoparticles comprising a distribution of sizes, wherein thepreparation does not comprise the nanoparticle of interest; (2)determining the size distribution of the preparation of nanoparticleswithout contacting the preparation with an optically detectable label;(3) contacting the preparation with an optically detectable label,wherein the optically detectable label comprises the surface area probeor volume probe in (a), wherein the surface area probe or volume probeinteracts with the nanoparticles stoichiometrically with respect tonanoparticle surface area or volume, respectively, whereby the opticalsignal from the optically detectable label is proportional to thesurface area or volume, respectively, of each nanoparticle in thepreparation; (4) detecting the optical signals obtained by (3), therebyobtaining the optical signal intensities of each nanoparticle in thepreparation; and (5) correlating the optical signal intensities obtainedin (4) with the size distribution determined in (2).
 16. The method ofclaim 14, wherein the preparation of nanoparticles comprises silicaparticles, wherein each silica particle comprises a lipid bilayer.
 17. Amethod of identifying and/or quantifying a nanoparticle of interest in asample using an optically detectable label, the method comprising: (a)contacting a nanoparticle of interest with an optically detectable labelcomprising a molecular marker-specific probe, wherein the opticallydetectable label interacts with a molecular marker associated with thenanoparticle of interest, whereby a nanoparticle of interest comprisingnanoparticle of interest-associated optically detectable label isobtained; (b) detecting the nanoparticle of interest-associatedoptically detectable label of (a), thereby obtaining an optical signalintensity; (c) obtaining a predetermined correlation between opticalsignal intensity and the number of molecular markers associated witheach nanoparticle of a preparation of nanoparticles, wherein: (i) thepreparation of nanoparticles is contacted with the optically detectablelabel used in (a); (ii) the optically detectable label interactsstoichiometrically with each of the nanoparticles of the preparation,whereby nanoparticles comprising nanoparticle-associated opticallydetectable label are obtained, wherein the optical signal from eachnanoparticle-associated optically detectable label is proportional tonumber of molecules of the molecular marker on the correspondingassociated nanoparticle; (iii) the optical signals of thenanoparticle-associated optically detectable labels of (ii) aredetected, thereby obtaining optical signal intensities corresponding tothe nanoparticle-associated optically detectable labels associated witheach nanoparticle of the preparation; and (iv) the optical signalintensity of each nanoparticle-associated optically detectable labelobtained in (iii) is correlated with the identity and/or quantity of itscorresponding associated nanoparticle; and (d) based on thepredetermined correlation obtained in (c), and based on the opticalsignal intensity obtained in (b), identifying and/or quantifying thenanoparticle of interest.
 18. The method of claim 17, wherein obtaininga correlation in (c) comprises: (1) obtaining a preparation ofnanoparticles comprising a distribution of different numbers ofmolecules of a molecular marker associated with each of thenanoparticles, wherein the molecular marker is the marker associatedwith the nanoparticle of interest in (a) and the preparation does notcomprise the nanoparticle of interest; (2) determining the numbers ofthe molecular markers in each nanoparticle of the preparation, withoutcontacting the preparation with an optically detectable label; (3)contacting the preparation with an optically detectable label, whereinthe optically detectable label comprises the molecular marker-specificprobe in (a), wherein the molecular marker-specific probe interacts withthe nanoparticles stoichiometrically with respect to the number ofmolecules of molecular marker associated with each nanoparticle of thempreparation, whereby the optical signal from the optically detectablelabel is proportional to the number of molecules of molecular markerassociated with each nanoparticle of the preparation; (4) detecting theoptical signals obtained by (3), thereby obtaining the optical signalintensities of each nanoparticle in the preparation; and (5) correlatingthe optical signal intensities obtained in (4) with the numbers of themolecular markers determined in (2).
 19. An optical standard particle,comprising a silica particle and a lipid bilayer in association with thesilica particle.
 20. A preparation comprising the optical standardparticle of claim 19, wherein the preparation comprises a plurality ofoptical standard particles comprising a range of sizes between about 10nm to about 900 nm.